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Monographs on Biochemistry 




ALCOHOLIC 
FERMENTATION 



ARTHUR HARDEN, PaD., D, 



(BERKELEY 
LIBRARY 

UNIVERSITY OF 
CALIFORNIA 

BIOLOGY LI1B. 



ri 



993 




MONOGRAPHS ON BIOCHEMISTRY 

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ALCOHOLIC 993 
FERMENTATION 



BY 



ARTHUR HARDEN, Pn.D., D.Sc., F.R.S. 

PROFESSOR OF BIOCHEMISTRY, LONDON UNIVERSITY 
HEAD OF THE BIOCHEMICAL DEPARTMENT, LISTER INSTITUTE, CHELSEA 




SECOND EDITION 



LONGMANS, GREEN AND CO. 

39 PATERNOSTER ROW, LONDON 

FOURTH AVENUE & 30TH STREET, NEW YORK 
BOMBAY, CALCUTTA, AND MADRAS! 

1914 



PREFACE. 

/ 

THE following chapters are based on courses of lectures 
delivered at the London University and the Royal Institution 
during 1909-1910. In them an account is given of the work 
done on alcoholic fermentation since Buchner's epoch-making 
discovery of zymase, only in so far as it appears to throw light 
on the nature of that phenomenon. Many interesting subjects, 
therefore, have perforce been left untouched, among them the 
problem of the formation of zymase in the cell, and the vexed 
question of the relation of alcoholic fermentation to the meta- 
bolic processes of the higher plants and animals. 

My thanks are due to the Council of the Royal Society, 
and to the Publishers of the " Journal of Physiology " for. 
permission to make use of blocks (Figs. 2, 4 and 7) which 
have appeared in their publications. 

A. H. 



PREFACE TO THE SECOND EDITION. 

IN the New Edition no change has been made in the scope 
of the work. The rapid progress of the subject has, 
however, rendered necessary many additions to the text and 
a considerable increase in the bibliography. 

A. H. 

May, 1914. 




Add to Lib. 



H3 



CONTENTS. 

CHAPTER PAGE 

I. HISTORICAL INTRODUCTION - i 

II. ZYMASE AND ITS PROPERTIES - 18 

III. THE FUNCTION OF PHOSPHATES IN ALCOHOLIC FERMENTATION 41 

IV. THE CO-ENZYME OF YEAST- JUICE - 59 

V. ACTION OF SOME INHIBITING AND ACCELERATING AGENTS ON 

THE ENZYMES OF YEAST- JUICE - 70 

VI. CARBOXYLASE - 81 

VII. THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION - - 85 

VIII. THE CHEMICAL CHANGES INVOLVED IN FERMENTATION - - 96 

IX. THE MECHANISM OF FERMENTATION - - 119 

BIBLIOGRAPHY - 136 

INDEX - 155 



600 



CHAPTER I. 

HISTORICAL INTRODUCTION. 

THE problem of alcoholic fermentation, of the origin and nature of that 
mysterious and apparently spontaneous change which converted the 
insipid juice of the grape into stimulating wine, seems to have exerted 
a fascination over the minds of natural philosophers from the very 
earliest times. No date can be assigned to the first observation of the 
phenomena of the process. History finds man in the possession of 
alcoholic liquors, and in the earliest chemical writings we find ferment- 
ation, as a familiar natural process, invoked to explain and illustrate the 
changes with which the science of those early days was concerned. 
Throughout the period of alchemy fermentation plays an important 
part ; it is, in fact, scarcely too much to say that the language of the 
alchemists and many of their ideas were founded on the phenomena of 
fermentation. The subtle change in properties permeating the whole 
mass of material, the frothing of the fermenting liquid, rendering evi- 
dent the vigour of the action, seemed to them the very emblems of the 
mysterious process by which the long sought for philosopher's stone 
was to convert the baser metals into gold. As chemical science 
emerged from the mists of alchemy, definite ideas about the nature 
of alcoholic fermentation and of putrefaction began to be formed. 
Fermentation was distinguished from other chemical changes in which 
gases were evolved, such as the action of acids on alkali carbonates 
(Sylvius de le Boe, 1659) ; the gas evolved was examined and termed 
gas vinorum, and was distinguished from the alcohol with which it 
had at first been confused (van Helmont, 1648); afterwards it was 
found that like the gas from potashes it was soluble in water (Wren, 
1664). The gaseous product of fermentation and putrefaction was 
identified by MacBride, in 1764, with the fixed air of Black, whilst 
Cavendish in 1766 showed that fixed air alone was evolved in alcoholic 
fermentation and that a mixture of this with inflammable air was pro- 
duced by putrefaction. In the meantime it had been recognised that 
only sweet liquors could be fermented (" Ubi notandum, nihil fer- 
mentare quod non sit dulce," Becher, 1682), and finally Cavendish 

I 



2 ALCOHOLIC FERMENTATION 

[1776] determined the proportion of fixed air obtainable from 
sugar by fermentation and found it to be 57 per cent. It gradually 
became recognised that fermentation might yield either spirituous or acid 
liquors, whilst putrefaction was thought to be an action of the same 
kind as fermentation, differing mainly in the character of the products 
(Becher). 

As regards the nature of the process very confused ideas at first 
prevailed, but in the time of the phlogistic chemists a definite theory of 
fermentation was proposed, first by Willis (1659) and afterwards by 
Stahl [1697], the fundamental idea of which survived the over- 
throw of the phlogistic system by Lavoisier and formed the foundation 
of the views of Liebig. To explain the spontaneous origin of ferment- 
ation and its propagation from one liquid to another, they supposed that 
the process consisted in a violent internal motion of the particles of the 
fermenting substance, set up by an aqueous liquid, whereby the com- 
bination of the essential constituents of this material was loosened and 
new particles formed, some of which were thrust out of the liquid (the 
carbon dioxide) and others retained in it (the alcohol). 

Stahl specifically states that a body in such a state of internal dis- 
quietude can very readily communicate the disturbance to another, 
which is itself at rest but is capable of undergoing a similar change, so 
that a putrefying or fermenting liquid can set another liquid in putre- 
faction or fermentation. 

Taking account of the gradual accumulation of fact and theory we 
find at the time of Lavoisier, from which the modern aspect of the 
problem dates, that Stahl's theoretical views were generally accepted. 
Alcoholic fermentation was known to require the presence of sugar and 
was thought to lead to the production of carbon dioxide, acetic acid, 
and alcohol. 

The composition of organic compounds was at that time not under- 
stood, and it was Lavoisier who established the fact that they consisted 
of carbon, hydrogen, and oxygen, and who made systematic analyses 
of the substances concerned in fermentation (1784-1789). Lavoisier 
[1789] applied the results of these analyses to the study of alcoholic 
fermentation, and by employing the principle which he regarded as the 
foundation of experimental chemistry, " that there is the same quantity 
of matter before and after the operation," he drew up an equation be- 
tween the quantities of carbon, hydrogen, and oxygen in the original 
sugar and in the resulting substances, alcohol, carbon dioxide, and 
acetic acid, showing that the products contained the whole matter of 
the sugar, and thus for the first time giving a clear view of the chemical 



HISTORICAL INTRODUCTION 3 

change which occurs in fermentation. The conclusion to which he 
came was, we now know, very nearly accurate, but the research must 
be regarded as one of those remarkable instances in which the genius 
of the investigator triumphs over experimental deficiencies, for the 
analytical numbers employed contained grave errors, and it was only by 
a fortunate compensation of these that a result so near the truth was 
attained. 

Lavoisier's equation or balance sheet was as follows : 

Carbon. Hydrogen. Oxygen. 

95-9 pounds of sugar (cane sugar) consist of . 26-8 77 61-4 
These yield : 

577 pounds of alcohol containing . . 167 9*6 31-4 

35-3 ,, carbon dioxide containing 9-9 25-4 

2*5 ,, acetic acid containing . 0*6 0*2 1*7 

Total contained in products . . . ! "C~ 27*2 9-8 58-5 

The true composition of the sugar used was carbon 40*4, hydrogen 
6-1, oxygen 49-4. 

Lavoisier expressed no view as to the agency by which ferment- 
ation was brought about, but came to a very definite and characteristic 
conclusion as to the chemical nature of the change. The sugar, which 
he regarded in harmony with his general views as an oxide, was split 
into two parts, one of which was oxidised at the expense of the other 
to form carbonic acid, whilst the other was deoxygenised in favour of 
the former to produce the combustible substance alcohol, " so that if it 
were possible to recombine these two substances, alcohol and carbonic 
acid, sugar would result ". 

From this point commences the modern study of the problem. 
Provided by the genius of Lavoisier with the assurance that the hitherto 
mysterious process of fermentation was to be ranked along with 
familiar chemical changes, and that it proceeded in harmony with the 
same quantitative laws as these simpler reactions, chemists were stimu- 
lated in their desire to penetrate further into the mysteries of the phe- 
nomenon, and the importance and interest of the problem attracted 
many workers. 

So important indeed did the matter appear to Lavoisier's country- 
men that in the year 8 of the French Republic (1800) a prize con- 
sisting of a gold medal, the value of which, expressed in terms of the 
newly introduced metric system, was that of one kilogram of gold 
was offered by the Institute for the best answer to the question : 
" What are the characteristics by which animal and vegetable substances 
which act as ferments can be distinguished from those which they are 
capable of fermenting? " 

I *- 



4 ALCOHOLIC FERMENTATION 

This valuable prize was again offered in 1802 but was never 
awarded, as the fund from which it was to be drawn was seques- 
trated from the Institute in 1 804. The first response to this stimulat- 
ing offer was an important memoir by citizen Thenard [1803], which 
provided many of the facts upon which Liebig subsequently based his 
views. Thenard combats the prevailing idea, first expressed by 
Fabroni (1787-1799), that fermentation is caused by the action of 
gluten derived from grain on starch and sugar, but is himself uncertain 
as to the actual nature of the ferment. He points out that all fer- 
menting liquids deposit a material resembling brewer's yeast, and he 
shows that this contains nitrogen, much of which is evolved as 
ammonia on distillation. His most important result is, however, 
that when yeast is used to ferment pure sugar, it undergoes a gradual 
change and is finally left as a white mass, much reduced in weight, 
which contains no nitrogen and is without action on sugar. Thenard, 
moreover, it is interesting to note, differs from Lavoisier, inasmuch 
as he ascribes the origin of some of the carbonic acid to the carbon 
of the ferment, an opinion which was still held in various degrees by 
many investigators (see Seguin, quoted by Thenard). 

Thenard's memoir was followed by a communication of funda- 
mental importance from Gay-Lussac [1810]. A process for preserv- 
ing food had been introduced by Appert, which consisted in placing 
the material in bottles, closing these very carefully and exposing 
them to the temperature of boiling water for some time. Gay- 
Lussac was struck by the fact that when such a bottle was opened 
fermentation or putrefaction set in rapidly. Analysis of the air left 
in such a sealed bottle showed that all the oxygen had been absorbed, 
and these facts led to the view that fermentation was set up by the 
action of oxygen on the fermentable material. Experiment appeared 
to confirm this in the most striking way. A bottle of preserved grape- 
juice was opened over mercury and part of its contents passed 
through the mercury into a bell-jar containing air, the remainder into 
a similar vessel free from air. In the presence of air fermentation set 
in at once, in the absence of air no fermentation whatever occurred. 
This connection between fermentation and the presence of air was 
established by numerous experiments and appeared incontestable. 
Fermentation, it was found, could be checked by boiling even after 
the addition of oxygen, and hence food could be preserved in free 
contact with the air, provided only that it was raised to the tempera- 
ture of boiling water at short intervals of time. Gay-Lussac's opinion 
was that the ferment was formed by the action of the oxygen on the 



HISTORICAL INTRODUCTION j 

liquid, and that the product of this action was altered by heat and 
rendered incapable of producing fermentation, as was also brewer's 
yeast, which, however, he regarded, on account of its insolubility, as 
different from the soluble ferment which initiated the change in the 
limpid grape-juice. Colin, on the other hand [1826], recognised that 
alcoholic fermentation by whatever substance it was started, resulted 
in the formation of an insoluble deposit more active than the 
original substance, and he suggested that this deposit might possibly 
in every case be of the same nature. 

So far no suspicion appears to have arisen in the minds of those 
who had occupied themselves with the study of fermentation that this 
change differed in any essential manner from many other reactions 
familiar to chemists. The origin and properties of the ferment were 
indeed remarkable and involved in obscurity, but the uncertainty re- 
garding this substance was no greater than that surrounding many, if 
not all, compounds of animal and vegetable origin. Although, how- 
ever, the purely chemical view as to the nature of yeast was generally 
recognised and adopted, isolated observations were not wanting which 
tended to show that yeast might be something more than a mere chemical 
reagent. As early as 1680 in letters to the Royal Society Leeuwen- 
hoek described the microscopic appearance of yeast of various origins 
as that of small, round, or oval particles, but no further progress seems 
to have been made in this direction for nearly a century and a half, 
when we find that Desmazieres [1826] examined the film formed 
on beer, figured the elongated cells of which it was composed, and 
described it under the name of Mycoderma Cerevisiae. He, however, 
regarded it rather as of animal than of vegetable origin, and does not 
appear to have connected the presence of these cells with the process 
of fermentation. 

Upon this long period during which yeast was regarded merely as 
a chemical compound there followed, as has so frequently occurred in 
similar cases, a sudden outburst of discovery. No less than three 
observers hit almost simultaneously upon the secret of fermentation and 
declared that yeast was a living organism. 

First among these in strict order of time was Cagniard-Latour 
[1838], who made a number of communications to the Academy 
and to the Soci6te Philomatique in 1835-6, the contents of which were 
collected in a paper presented to the Academy of Sciences on 12 June, 
1837, and published in 1838. The observations upon which this 
memoir was based were almost exclusively microscopical. Yeast was 
recognised as consisting of spherical particles, which were capable of 



6 ALCOHOLIC FERMENTATION 

reproduction by budding but incapable of motion, and it was therefore 
regarded as a living organism probably belonging to the vegetable 
kingdom. Alcoholic fermentation was observed to depend on the 
presence of living yeast cells, and was attributed to some effect of their 
vegetative life (quelque effet de leur vegetation). It was also noticed 
that yeast was not deprived of its fermenting power by exposure to 
the temperature of solid carbonic acid, a sample of which was supplied 
to Cagniard-Latour by Thilorier, who had only recently prepared it for 
the first time. 

Theodor Schwann [1837], whose researches were quite inde- 
pendent of those of Cagniard-Latour, approached the problem from an 
entirely different point of view. During the year 1836 Franz Schulze 
[1836] published a research on the subject of spontaneous gener- 
ation, in which he proved that when a solution containing animal or 
vegetable matter was boiled, no putrefaction set in provided that all 
air which was allowed to have access to the liquid was previously 
passed through strong sulphuric acid. Schwann performed a very 
similar experiment by which he showed that this same result, the 
absence of putrefaction, was attained by heating all air which came into 
contact with the boiled liquid. Wishing to show that other processes 
in which air took part were not affected by the air being heated, he 
made experiments with fermenting liquids and found, contrary to his 
expectation, that a liquid capable of undergoing vinous fermentation 
and containing yeast did not undergo this change after it had been 
boiled, provided that, as in the case of his previous experiments, 
only air which had been heated was allowed to come into contact 
with it. 

Schwann's experiments on the prevention of putrefaction were un- 
exceptionable and quite decisive. The analogous experiments dealing 
with alcoholic fermentation were not quite so satisfactory. Yeast was 
added to a solution of cane sugar, the flask containing the mixture 
placed in boiling water for ten minutes, and then inverted over mer- 
cury. About one-third of the liquid was then displaced by air and the 
flasks corked and kept inverted at air temperature. In two flasks the 
air introduced was ordinary atmospheric air, and in these flasks fermen- 
tation set in after about four to six weeks. Into the other two flasks 
air which had been heated was led, and in these no fermentation 
occurred. As described, the experiment is quite satisfactory, but 
Schwann found on repetition that the results were irregular. Some- 
times all the flasks showed fermentation, sometimes none of them. 
This was correctly ascribed to the experimental difficulties, but none 



HISTORICAL INTRODUCTION 7 

the less served as a point of attack for hostile and damaging criticism 
at the hands of Berzelius (p. 8). 

The origin of putrefaction was definitely attributed by Schwann to 
the presence of living germs in the air, and the similarity of the result 
obtained with yeast suggested the idea that alcoholic fermentation was 
also brought about by a living organism, a conception which was at 
once confirmed by a microscopical examination of a fermenting 
liquid. The phenomena observed under the microscope were similar 
to those noted by Cagniard-Latour, and in accordance with these 
observations alcoholic fermentation was attributed to the development 
of a living organism, the fermentative function of which was found to 
be destroyed by potassium arsenite and not by extract of Nux vomica, 
so that the organism was regarded rather as of vegetable than of 
animal nature. This plant received the name of " Zuckerpilz " or 
sugar fungus (which has been perpetuated in the generic term Sac- 
charomyces). Alcoholic fermentation was explained as " the decom- 
position brought about by this sugar fungus removing from the sugar 
and a nitrogenous substance the materials necessary for its growth and 
nourishment, whilst the remaining elements of these compounds, which 
were not taken up by the plant, combined chiefly to form alcohol ". 

Kiitzing's memoir, the third of the trio [1837], also dates from 
1837, and his opinions, like those of Cagniard-Latour, are founded on 
microscopical observations. He recognises yeast as a vegetable 
organism and accurately describes its appearance. Alcoholic ferment- 
ation depends on the formation of yeast, which is produced when the 
necessary elements and the proper conditions are present and then 
propagates itself. The action on the liquid thus increases and the 
constituents not required to form the organism combine to form un- 
organised substances, the carbonic acid and alcohol. " It is obvious," 
says Kiitzing, in a passage which roused the sarcasm of Berzelius, 
" that chemists must now strike yeast off the roll of chemical com- 
pounds, since it is not a compound but an organised body, an 
organism." 

These three papers, which were published almost simultaneously, 
were received at first with incredulity. Berzelius, at that time the 
arbiter and dictator of the chemical world, reviewed them all in his 
" Jahresbericht " for 1839 [1839] with impartial scorn. The micro- 
scopical evidence was denied all value, and yeast was no more to be 
regarded as an organism than was a precipitate of alumina. Schwann's 
experiment (p. 6) was criticised on the ground that the fermenting 
power of the added yeast had been only partially destroyed in the 



8 ALCOHOLIC FERMENTATION 

flasks in which fermentation ensued, completely in those which re- 
mained unchanged, the admission of heated or unheated air being 
indifferent, a criticism to some extent justified by Schwann's statement, 
already quoted, of the uncertain result of the experiment 

Berzelius himself regarded fermentation as being brought about 
by the yeast by virtue of that catalytic force, which he had supposed 
to intervene in so many reactions, both between substances of mineral 
and of animal and vegetable origin [1836], and which enabled " bodies, 
by their mere presence, and not by their affinity, to arouse affinities 
ordinarily quiescent at the temperature of the experiment, so that the 
elements of a compound body arrange themselves in some different 
way, by which a greater degree of electro-chemical neutralisation is 
attained". 

To the scorn of Berzelius was soon added the sarcasm of Wohler 
and Liebig [1839]. Stimulated in part by the publications of the 
three authors already mentioned, and in part by the report of Turpin 
[1839], who at the request of the Academy of Sciences had satisfied 
himself by observation of the accuracy of Cagniard-Latour's con- 
clusions, Wohler prepared an elaborate skit on the subject, which he 
sent to Liebig, to whom it appealed so strongly that he added some 
touches of his own and published it in the " Annalen," following im- 
mediately upon a translation of Turpin's paper. Yeast was here des- 
cribed with a considerable degree of anatomical realism as consisting 
of eggs which developed into minute animals, shaped like a distilling 
apparatus, by which the sugar was taken in as food and digested into 
carbonic acid and alcohol, which were separately excreted, the whole 
process being easily followed under the miscroscope. 

Close upon this pleasantry followed a serious and important com- 
munication from Liebig [1839], in which the nature of fermentation, 
putrefaction, and decay was exhaustively discussed. Liebig did not ad- 
mit that these phenomena were caused by living organisms, nor did he 
attribute them like Berzelius to the catalytic action of a substance which 
itself survived the reaction unchanged. As regards alcoholic fermenta- 
tion, Liebig's chief arguments may be briefly summarised. As the 
result of alcoholic fermentation, the whole of the carbon of the sugar 
reappears in the alcohol and carbon dioxide formed. This change is 
brought about by a body termed the ferment, which is formed as the result 
of a change set up by the access of air to plant juices containing sugar, 
and which contains all the nitrogen of the nitrogenous constituents of the 
juice. This ferment is a substance remarkably susceptible of change, 
which undergoes an uninterrupted and progressive metamorphosis, of 



HISTORICAL INTRODUCTION 9 

the nature of putrefaction or decay, and produces the fermentation of 
the sugar as a consequence of the transformation which it is itself 
undergoing. 

The decomposition of the sugar is therefore due to a condition of 
instability transferred to it from the unstable and changing ferment, and 
only continues so long as the decomposition of the ferment proceeds. 
This communication of instability from one substance undergoing 
chemical change to another is the basis of Liebig's conception, and is 
illustrated by a number of chemical analogies, one of which will suffice 
to explain his meaning. Platinum is itself incapable of decomposing 
nitric acid and dissolving in it ; silver, on the other hand, possesses this 
power. When platinum is alloyed with silver, the whole mass dissolves 
in nitric acid, the power possessed by the silver being transferred to 
the platinum. In like manner the condition of active decomposition 
of the ferment is transferred to the sugar, which by itself is quite stable. 
The central idea is that of Stahl (p. 2) which was thus reintroduced 
into scientific thought. 

In a pure sugar solution the decomposition of the ferment soon 
comes to an end and fermentation then ceases. In beer wort or veget- 
able juices, on the other hand, more ferment is continually formed in 
the manner already described from the nitrogenous constituents of the 
juice, and hence the sugar is completely fermented away and un- 
exhausted ferment left behind. Liebig's views were reiterated in his 
celebrated "Chemische Briefe," and became the generally accepted 
doctrine of chemists. There seems little doubt that both Berzelius 
and Liebig in their scornful rejection of the results of Cagniard-Latour, 
Schwann and Kiitzing, were influenced, perhaps almost unconsciously, 
by a desire to avoid seeing an important chemical change relegated to 
the domain of that vital force from beneath the sway of which a large 
part of organic chemistry had just been rescued by Wohler's brilliant 
synthetical production of urea and by the less recognised synthesis of 
alcohol by Hennell (see on this point Ahrens [1902]). A strong 
body of evidence, however, gradually accumulated in favour of the 
vegetable nature of yeast, so that it may be said that by 1848 a 
powerful minority adhered to the views of Cagniard-Latour, Schwann, 
and Kiitzing [see Schrohe, 1904, p. 218, and compare Buchner, 
1904]. Among these must be included Berzelius [1848], who had 
so forcibly repudiated the idea only ten years before, whereas Liebig 
in the 1851 edition of his letters does not mention the fact that yeast 
is a living organism (Letter XV). 

The recognition of the vegetable nature of yeast, however, by no 



io ALCOHOLIC FERMENTATION 

means disproved Liebig's view of the nature of the change by which 
sugar was converted into carbon dioxide and alcohol, as was carefully 
pointed out by Schlossberger [1844] in a research on the nature of 
yeast, carried out in Liebig's laboratory but without decisive results. 

Mitscherlich was also convinced of the vegetable character of yeast, 
and showed [1841] that when yeast was placed in a glass tube 
closed by parchment and plunged into sugar solution, the sugar 
entered the glass tube and was there fermented, but was not fermented 
outside the tube. He regarded this as a proof that fermentation only 
occurred at the surface of the yeast cells, and explained the process by 
contact action in the sense of the catalytic action of Berzelius, rather 
than by Liebig's transference of molecular instability. Similar results 
were obtained with an animal membrane by Helmholtz [1843], 
who also expressed his conviction that yeast was a vegetable or- 
ganism. 

In 1854 Schroder and von Dusch [1854, 1859, 1861] strongly rein- 
forced the evidence in favour of this view by succeeding in preventing 
the putrefaction and fermentation of many boiled organic liquids by 
the simple process of filtering all air which had access to them through 
cotton-wool. These experiments, which were continued until 1861, 
led to the conclusion that the spontaneous alcoholic fermentation of 
liquids was due to living germs carried by the air, and that when the 
air was passed through the cotton-wool these germs were held back. 

At the middle of the nineteenth century opinions with regard to 
alcoholic fermentation, notwithstanding all that had been done, were 
still divided. On the one hand Liebig's theory of fermentation was 
widely held and taught. Gerhardt, for example, as late as 1856 in the 
article on fermentation in his treatise on organic chemistry [1856], 
gives entire support to Liebig's views, and his treatment of the matter af- 
fords an interesting glimpse of the arguments which were then held to be 
decisive. The grounds on which he rejects the conclusions of Schwann 
and the other investigators who shared the belief in the vegetable nature 
of yeast are that, although in some cases animal and vegetable matter 
and infusions can be preserved from change by the methods described 
by these authors, in others they cannot, a striking case being that of 
milk, which even after being boiled becomes sour even in filtered air, 
and this without showing any trace of living organisms. The action 
of heat, sulphuric acid, and filtration on the air is to remove, or destroy, 
not living organisms but particles of decomposing matter, that is to 
say, ferments which would add their activity to that of the oxygen of 
the air. Moreover, many ferments, as for example diastase, act with- 



HISTORICAL INTRODUCTION it 

out producing any insoluble deposit whatever which can be regarded 
as an organism. 

" Evidemment," he concludes, " la theorie de M. Liebig explique 
seule tous les phenomenes de la maniere la plus complete et la plus 
logique ; c'est a elle que tous les bons esprits ne peuvent manquer de 
se rallier." 

On the other hand it was held by many to have been shown that 
Liebig' s view of the origin of yeast by the action of the air on a veget- 
able infusion was erroneous, and that fermentation only arose when 
the air transferred to the liquid an active agent which could be removed 
from it by sulphuric acid (Schulze), by heat (Schwann), and by cotton- 
wool (Schroder and von Dusch). Accompanying alcoholic ferment- 
ation there was a development of a living organism, the yeast, and 
fermentation was believed, without any very strict proof, to be a phe- 
nomenon due to the life and vegetation of this organism. This doctrine 
seems indeed [Schrohe, 1904] to have been widely taught in Ger- 
many from 1840-56, and to have established itself in the practice of 
the fermentation industries. 

In 1857 commenced the classical researches of Pasteur which finally 
decided the question as to the origin and functions of yeast and led 
him to the conclusion that " alcoholic fermentation is an act correlated 
with the life and organisation of the yeast cells, not with the death or 
putrefaction of the cells, any more than it is a phenomenon of contact, 
in which case the transformation of sugar would be accomplished in 
presence of the ferment without yielding up to it or taking from it 
anything" [1860]. It is impossible here to enter in detail into 
Pasteur's experiments on this subject, or indeed to do more than indi- 
cate the general lines of his investigation. His starting-point was the 
lactic acid fermentation. 

The organism to which this change was due had hitherto escaped 
detection, and as we have seen the spontaneous lactic fermentation of 
milk was one of the phenomena adduced by Gerhardt (p. 10) in favour of 
Liebig's views. Pasteur [1857] discovered the lactic acid produc- 
ing organism and convinced himself that it was in fact a living organ- 
ism and the active cause of the production of lactic acid. One of the 
chief buttresses of Liebig's theory was thus removed, and Pasteur next 
proceeded to apply the same method and reasoning to alcoholic ferment- 
ation. Liebig's theory of the origin of yeast by the action of the oxygen 
of the air on the nitrogenous matter of the fermentable liquid was con- 
clusively and strikingly disproved by the brilliant device of producing 
a crop of yeast in a liquid medium containing only comparatively 



i2 ALCOHOLIC FERMENTATION 

simple substances of known composition sugar, ammonium tartrate and 
mineral phosphate. Here there was obviously present in the original 
medium no matter which could be put into a state of putrefaction by 
contact with oxygen and extend its instability to the sugar. Any such 
material must first be formed by the vital processes of the yeast In 
the next place Pasteur showed by careful analyses and estimations that, 
whenever fermentation occurred, growth and multiplication of yeast ac- 
companied the phenomenon. The sugar, he proved, was not completely 
decomposed into carbon dioxide and alcohol, as had been assumed by 
Liebig (p. 8). A balance-sheet of materials and products was con- 
structed which showed that the alcohol and carbon dioxide formed 
amounted only to about 95 per cent, of the invert sugar fermented, the 
difference being made up by glycerol, succinic acid, cellulose, and other 
substances [1860, p. 347]. In every case of fermentation, even 
when a paste of yeast was added to a solution of pure cane sugar in 
water, the yeast was found by quantitative measurements to have taken 
something from the sugar. This " something " was indeterminate in 
character, but, including the whole of the extractives which had passed 
from the yeast cells into the surrounding liquid, it amounted to as much 
as 1-63 per cent, of the weight of the sugar fermented [1860, 

P- 344]- 

Pasteur was therefore led to consider fermentation as a physiolog- 
ical process accompanying the life of the yeast. His conclusions were 
couched in unmistakable words : " The chemical act of fermentation is 
essentially a phenomenon correlative with a vital act, commencing and 
ceasing with the latter. I am of opinion that alcoholic fermentation 
never occurs without simultaneous organisation, development, multi- 
plication of cells, or the continued life of cells already formed. The 
results expressed in this memoir seem to me to be completely opposed 
to the opinions of Liebig and Berzelius. If I am asked in what con- 
sists the chemical act whereby the sugar is decomposed and what is 
its real cause, I reply that I am completely ignorant of it. 

" Ought we to say that the yeast feeds on sugar and excretes alcohol 
and carbonic acid ? Or should we rather maintain that yeast in its 
development produces some substance of the nature of a pepsin, which 
acts upon the sugar and then disappears, for no such substance is found 
in fermented liquids ? I have nothing to reply to these hypotheses. 
I neither admit them nor reject them, and wish only to restrain myself 
from going beyond the facts. And the facts tell me simply that all 
true fermentations are correlative with physiological phenomena." 

Liebig felt to the full the weight of Pasteur's criticisms ; his reply 



HISTORICAL INTRODUCTION 13 

was long delayed [1870], and, according to his biographer, 
Volhard [1909], caused him much anxiety. In it he admits the 
vegetable nature of yeast, but does not regard Pasteur's conclusion 
as in any way a solution of the problem of the nature of alcoholic 
fermentation. Pasteur's "physiological act" is for Liebig the very 
phenomenon which requires explanation, and which he still maintains 
can be explained by his original theory of communicated instability. 
On some of Pasteur's results, notably the very important one of the 
cultivation of yeast in a synthetic medium, he casts grave doubt, 
whilst he explains the production of glycerol and succinic acid as due 
to independent reactions. The phenomenon of fermentation is still for 
him one which accompanies the decomposition of the constituents 
of the cell, rather than their building up by vegetative growth. 
" When the fungus ceases to grow, the bond which holds together 
the constituents of the cell contents is relaxed, and it is the motion 
which is thus set up in them which is the means by which the yeast 
cells are enabled to bring about a displacement or decomposition 
of the elements of sugar or other organic molecules." Pasteur replied 
in a brief and unanswerable note [1872]. All his attention was 
concentrated on the one question of the production of yeast in a 
synthetic medium, which he recognised as fundamental. The validity 
of this experiment he emphatically reaffirmed, and finally undertook, 
from materials supplied by Liebig himself, to produce as much yeast as 
could be reasonably desired. This challenge was never taken up, and 
this communication formed the last word of the controversy. Pasteur 
had at this time firmly established his thesis, no fermentation without 
life, both for alcoholic fermentation and for those other fermentations 
which are produced by bacteria, and had put upon a sound and per- 
manent basis the conclusions drawn by Schulze, Cagniard-Latour, 
Schwann, and Kiitzing from their early experiments. It became 
generally recognised that putrefaction and other fermentative changes 
were due to specific organisms, which produced them in the exercise 
of their vital functions. 

Pasteur subsequently [1875] came to the conclusion that fer- 
mentation was the result of life without oxygen, the cells being able, 
in the absence of free oxygen, to avail themselves of the energy liber- 
ated by the decomposition of substances containing combined oxygen. 
This view, which did not involve any alteration of Pasteur's original 
thesis but was an attempt to explain the physiological origin and 
function of fermentation, gave rise to a prolonged controversy, which 
cannot be further discussed in these pages. 



I 4 ALCOHOLIC FERMENTATION 

Nevertheless, Liebig's desire to penetrate more deeply into the 
nature of the process of fermentation remained in many minds, and 
numerous endeavours were made to obtain further insight into the 
problem. In spite of an entire lack of direct experimental proof, the 
conception that alcoholic fermentation was due to the chemical action 
of some substance elaborated by the cell and not directly to the 
vital processes of the cell as a whole found strenuous supporters even 
among those who were convinced of the vegetable character of yeast. 
As early as 1833 diastase, discovered still earlier by Kirchhoff and 
Dubrunfaut, had been extracted by means of water from germinating 
barley and precipitated by alcohol as a white powder, the solution of 
which was capable of converting starch into sugar, but lost this power 
when heated [Payen and Persoz, 1833]. Basing his ideas in part 
upon the behaviour of this substance, Moritz Traube [1858] enun- 
ciated in the clearest possible manner the theory that all ferment- 
ations produced by living organisms are caused by ferments, which are 
definite chemical substances produced in the cells of the organism. He 
regarded these substances as being closely related to the proteins and 
considered that their function was to transfer the oxygen and hy- 
drogen of water to different parts of the molecule of the fermentable 
substance and thus bring about that apparent intramolecular oxi- 
dation and reduction which is so characteristic of fermentative 
change and had arrested the attention of Lavoisier and, long after 
him, of Liebig. 

Traube's main thesis, that fermentation is caused by definite fer- 
ments or enzymes, attracted much attention, and received fresh sup- 
port from the separation of invertase in 1 860 from an extract of yeast 
by Berthelot, and from the advocacy and authority of this great 
countryman of Pasteur, who definitely expressed his opinion that 
insoluble ferments existed which could not be separated from the 
tissues of the organism, and further, that the organism could not itself 
be regarded as the ferment, but only as the producer of the ferment 
[1857, 1860]. Hoppe-Seyler [1876] also supported the enzyme 
theory of fermentation, but differed in some respects from Traube 
as to the exact function of the ferment [see Traube, 1877; Hoppe- 
Seyler, 1877]. 

Direct experimental evidence was, however, still wanting, and 
Pasteur's reiterated assertion [1875] that all fermentation phenomena 
were manifestations of the life of the organism remained uncontroverted 
by experience. 

Numerous and repeated direct experimental attacks had been made 



HISTORICAL INTRODUCTION 15 

from time to time upon the problem of the existence of a fermentation 
enzyme, but all had yielded negative or unreliable results. 

As early as 1846 a bold attempt had been made by Liidersdorff 
[1846] to ascertain whether fermentation was or was not bound up 
with the life of the yeast by grinding yeast and examining the ground 
mass. A single gram of yeast was thoroughly ground, the process 
lasting for an hour, and the product was tested with sugar solution. 
Not a single bubble of gas was evolved. A similar result was 
obtained in a repetition of the experiment by Schmidt in Liebig's 
laboratory [1847], the grinding being continued in this case for six 
hours, but the natural conclusion that living yeast was essential for 
fermentation was not accepted, on the ground that during the' lengthy 
process of trituration in contact with air the yeast had become altered 
and now no longer possessed the power of producing alcoholic fer- 
mentation, but instead had acquired that of changing sugar into lactic 
acid [see Gerhardt, 1856, p. 545]. 

Similar experiments made in 1871 by Marie von Manassei'n [1872, 
J 897], in which yeast was ground for six to fifteen hours with 
powdered rock crystal, yielded products which fermented sugar, but 
they contained unbroken yeast cells, so that the results obtained could 
not be considered decisive [Buchner and Rapp, 1898, i], although 
Frau von Manassei'n herself drew from them and from others in which 
sugar solution was treated with heated yeast, but not under aseptic con- 
ditions, the conclusion that living yeast cells were not necessary for 
fermentation. 

Quite unsuccessful were also the attempts made to accomplish the 
separation of fermentation from the living cell by Adolf Mayer [1879, 
p. 66], and, as we learn from Roux, by Pasteur himself, grinding, 
freezing, and plasmolysing the cells, having in his hands proved alike 
in vain. Extraction by glycerol or water, a method by which many 
enzymes can be obtained in solution, gave no better results [Nageli 
and Loew, 1878], and the enzyme theory of alcoholic fermentation 
appeared quite unjustified by experiment. 

Having convinced himself of this, Nageli [1879] suggested a 
new explanation of the facts based on molecular-physical grounds. 
According to this view, which unites in itself some of the conceptions 
of Liebig, Pasteur, and Traube, fermentation is the transference of a 
state of motion from the molecules, atomic groups, and atoms of the 
compounds constituting the living plasma of the cell to the ferment- 
able material, whereby the equilibrium existing in the molecules of the 
latter is disturbed and decomposition ensues [1879, p. 29]. 



16 ALCOHOLIC FERMENTATION 

This somewhat complex idea, whilst including, as did Liebig's 
theory, Stahl's fundamental conception of a transmission of a state of 
motion, satisfies Pasteur's contention that fermentation cannot occur 
without life, and at the same time explains the specific action of differ- 
ent organisms by differences in the constitution of their cell contents. 
The really essential part of Nageli's theory consisted in the limitation 
of the power of transference of molecular motion to the living plasma, 
by which the failure of all attempts to separate the power of ferment- 
ation from the living cell was explained. This was the special phe- 
nomenon which required explanation ; to account for this the theory 
was devised, and when this was experimentally disproved, the theory 
lost all significance. 

For nearly twenty years no further progress was made, and then 
in 1 897 the question which had aroused so much discussion and con- 
jecture, and had given rise to so much experimental work, was finally 
answered by Eduard Buchner, who succeeded in preparing from yeast 
a liquid which, in the complete absence of cells, was capable of effecting 
the resolution of sugar into carbon dioxide and alcohol [1897, i]. 

In the light of this discovery the contribution to the truth made 
by each of the great protagonists in the prolonged discussion on the 
problem of alcoholic fermentation can be discerned with some degree 
of clearness. Liebig's main contention that fermentation was essen- 
tially a chemical act was correct, although his explanation of the nature 
of this act was inaccurate. Pasteur, in so far as he considered the act 
of fermentation as indissolubly connected with the life of the organism, 
was shown to be in error, but the function of the organism has only 
been restricted by a single stage, the active enzyme of alcoholic fer- 
mentation has so far only been observed as the product of the living 
cell. Nearest of all to the truth was Traube, who in 1858 enunciated 
the theorem, which was only proved for alcoholic fermentation in 
1897, that all fermentations produced by living organisms are due to 
ferments secreted by the cells. 

Buchner's discovery of zymase has introduced a new experimental 
method by means of which the problem of alcoholic fermentation can 
be attacked, and the result has been that since 1897 a considerable 
amount of information has been gained with regard to the nature and 
conditions of action of the enzymes of the yeast cell. It has been 
found that the machinery of fermentation is much more complex than 
had been surmised. The enzyme zymase, which is essential for fer- 
mentation, cannot of itself bring about the alcoholic fermentation of 
sugar, but is dependent on the presence of a second substance, termed, for 



HISTORICAL INTRODUCTION i; 

want of a more reasonable name, the co-enzyme. The chemical nature 
and function of this mysterious coadjutor are still unknown, but as it 
withstands the temperature of boiling water and is dialysable, it is 
probably more simple in constitution than the enzyme. This, however, 
is not all ; for the decomposition of sugar a phosphate is also indis- 
pensable. It appears that in yeast-juice, and therefore also most pro- 
bably in the yeast cell, the phosphorus present takes an active part in 
fermentation and goes through a remarkable cycle of changes. The 
breakdown of sugar into alcohol and carbon dioxide is accompanied 
by the formation of a complex hexosephosphate, and the phosphate 
is split off from this compound and thus again rendered available 
for action by means of a special enzyme, termed hexosephosphatase. 
In addition to this complex of ferments, the cell also possesses special 
enzymes by which the zymase and the co-enzyme can be destroyed, and, 
further, at least one substance, known as an anti-enzyme, which 
directly checks this destructive action. It seems probable, moreover, 
that the decomposition of the sugar molecule takes place in stages, 
although much doubt yet exists as to the nature of these. 

At the present moment the subject remains one of the most 
interesting in the whole field of biological chemistry, the limited 
degree of insight which has already been gained into the marvellous 
complexity of the cell lending additional zest to the attempt to pene- 
trate the darkness which shrouds the still hidden mysteries. 



CHAPTER II. 

ZYMASE AND ITS PROPERTIES. 

Discovery of Zymase. 

THE history of Buchner's discovery is of great interest [Gruber, 1908 ; 
Hahn, 1908]. As early as 1893 Hans and Eduard Buchner found 
that the cells of even the smallest micro-organism could be broken 
by being ground with sand [Buchner, E. and H., and Hahn, 1903, 
p. 20], and in 1896 the same process was applied by these two inves- 
tigators to yeast, with the object of obtaining a preparation for 
therapeutic purposes. Difficulties arose in the separation of the cell 
contents from the ground-up mixture of cell membranes, unbroken 
cells, and sand, but these were overcome by carrying out the sugges- 
tion of Martin Hahn (at that time assistant to Hans Buchner) that 
kieselguhr should be added and the liquid squeezed out by means of 
a hydraulic press [Buchner, E. and H., and Hahn, 1903, p. 58]. The 
yeast-juice thus obtained was, in the first instance, employed for animal 
experiments, but underwent change very rapidly. The ordinary anti- 
septics were found to be unsuitable, and hence sugar was added as a 
preservative, and it was the marked action of the juice upon this added 
cane sugar that drew Eduard Buchner's attention to the fact that 
fermentation was proceeding in the absence of yeast-cells. 

As in the case of so many discoveries, the new phenomenon was 
brought to light, apparently by chance, as the result of an investigation 
directed to quite other ends, but fortunately fell under the eye of an 
observer possessed of the genius which enabled him to realise its 
importance and give to it the true interpretation. 

In his first papers [1897, I, 2 ; 1898], Buchner established the 
following facts : (i) yeast-juice free from cells is capable of producing 
the alcoholic fermentation of glucose, fructose, cane sugar, and 
maltose ; (2) the fermenting power of the juice is neither destroyed 
by the addition 6f chloroform, benzene, or sodium arsenite [Hans 
Buchner, 1897], by filtration through a Berkefeld filter, by evaporation 
to dryness at 30 to 35, nor by precipitation with alcohol; (3) the 
fermenting power is completely destroyed when the liquid is heated 
to 50. 

18 



ZYMASE AND ITS PROPERTIES 19 

From these facts he drew the conclusion "that the production of 
alcoholic fermentation does not require so complicated an apparatus 
as the yeast cell, and that the fermentative power of yeast-juice is due 
to the presence of a dissolved substance". To this active substance 
he gave the name of zymase. 

Buchner's discovery was not received without some hesitation. A 
number of investigators prepared yeast-juice, but failed to obtain an 
active product [Will, 1897; Delbruck, 1897; Martin and Chapman, 
1 898 ; Reynolds Green, 1 897 ; Lintrier, 1 899]. A more accurate know- 
ledge of the necessary conditions and of the properties of yeast-juice, 
however, led to more successful results [Will, 1 898 ; Reynolds Green, 
1898; Lange, 1898], and it was soon established that, given suitable 
yeast, an active preparation could be readily procured by Buchner's 
method. Criticism was then directed to the effect of the admitted 
presence of a certain number of micro-organisms in yeast-juice [Staven- 
hagen, 1897], but Buchner [Buchner and Rapp, 1897] was able to show 
by experiments in the presence of antiseptics and with juice filtered 
through a Chamberland candle that the fermentation was not due 
to living organisms of any kind. 

The most weighty criticism of Buchner's conclusion consisted in an 
attempt to show that the properties of yeast-juice might be due to the 
presence, suspended in it, of fragments of living protoplasm, which, 
although severed from their original surroundings in the cell, might 
retain for some time the power of producing alcoholic fermentation. 
This, it will be seen, was an endeavour to extend Nageli's theory to 
include in it the newly discovered fact. 

In favour of this view were adduced the similarity between the 
effects of many antiseptics on living yeast and on the juice, the 
ephemeral nature of the fermenting agent present in the juice, the effect 
of dilution with water, and the phenomenon of auto fermentation which 
is exhibited by the juice in the absence of added sugar [Abeles, 1898 ; 
v. Kupffer, 1897; v. Voit, 1897; Wehmer, 1898; Neumeister, 1897; 
Macfadyen, Morris, and Rowland, 1900; Bokorny, 1906; Fischer, 
1903; Beijerinck, 1897, 1900; Wroblewski, 1899, 1901]. 

A brief general description of the actual properties of yeast-juice 
and of the phenomena of fermentation by its means is sufficient to 
show the great improbability of this view. 

The juice prepared by Buchner's method forms a somewhat viscous 
opalescent brownish-yellow liquid, which is usually faintly acid in 
reaction [compare Ahrens, 1900] and almost optically inactive. It 
has a specific gravity of 1*03 to I'o6, contains 8'5 to 14 per cent. 

2 * 



20 ALCOHOLIC FERMENTATION 

of dissolved solids, and leaves an ash amounting to i -4 to 2 per cent. 
About 07 to 17 per cent, of nitrogen is present, nearly all in the form 
of protein, which coagulates to a thick white mass when the juice is 
heated. 

A powerful digestive enzyme of the type of trypsin is also present, so 
that when the juice is preserved its albumin undergoes digestion at a rate 
which depends on the temperature [Hahn, 1 898 ; Geret and Hahn, 1 898, 
1,2; 1900 ; Buchner, E. and H., and Hahn, 1903, pp. 287-340], and is 
converted into a mixture of bases and amino-acids. After about six days 
at 37, or i o to 14 days at the ordinary temperature, the digestion is so 
complete that no coagulation occurs when the juice is boiled. As this pro- 
teoclastic enzyme, like the alcoholic enzyme, cannot be extracted from 
the living cells, it is termed yeast endotrypsin or endotryptase. Fresh 
yeast-juice produces a slow fermentation of sugar, which lasts for forty- 
eight to ninety-six hours at 25 to 30, about a week at the ordinary 
temperature, and then ceases, owing, not to exhaustion of the sugar, 
but to the disappearance of the fermenting agent. When the juice 
is preserved or incubated in the absence of a fermentable sugar this 
disappearance occurs considerably sooner, so that even after standing 
for a single day at room temperature, or two days at o, no fermen- 
tation may occur when sugar is added. The reason for this behaviour 
has not been definitely ascertained. As will be seen later on (p. 64) 
the phenomenon is a complex one, but the disappearance of the 
enzyme was originally ascribed by Buchner to the digestive action 
upon it of the endotrypsin of the juice [1897, 2], and no better explana- 
tion has yet been found. Confirmation of this view is afforded by the 
fact that the addition of a tryptic enzyme of animal origin greatly 
hastens the disappearance of the alcoholic enzyme [Buchner, E. and H., 
and Hahn, 1903, p. 126], and that some substances which hinder the 
tryptic action favour fermentation [Harden, 1903]. The amount of 
fermentation produced is almost unaffected by the presence of such 
antiseptics as chloroform or toluene, although some others, such as 
arsenites and fluorides, decrease it when added in comparatively high 
concentrations, and it is only slightly diminished by dilution with 
three or four volumes of sugar solution, somewhat more considerably 
by dilution with water. When it is filtered through a Chamberland 
filter the first portions of the filtrate are capable of bringing about 
fermentation, but the fermenting power diminishes in the succeeding 
portions and finally disappears. The juice can be spun in a centrifugal 
machine without being in any way altered, and no separation into 
more or less active layers takes place under these conditions. 



ZYMASE AND ITS PROPERTIES 21 

The amorphous powder obtained by drying the precipitate produced 
when the juice is added to a mixture of alcohol and ether is also cap- 
able of producing fermentation, and the process of precipitation may 
be repeated without seriously diminishing the fermenting power of the 
product. 

These facts clearly show that the various phenomena adduced by 
the supporters of the theory of protoplasmic fragments are quite con- 
sistent with the presence of a dissolved enzyme as the active agent of 
the juice, and at the same time that the properties demanded of the 
living fragments of protoplasm to which fermentation is ascribed are 
such as cannot be reconciled with our knowledge of living matter. If 
living protoplasm is the cause of alcoholic fermentation by yeast-juice, 
a new conception of life will be necessary; the properties of the 
postulated fragments of protoplasm must be so different from those 
which the protoplasm of the living cell possesses as to deprive the 
theory of all real value [Buchner, 1900, 2 ; Buchner, E. and H., and 
Hahn, 1903, p. 33]. 

Further and very convincing evidence against the protoplasm theory 
is afforded by the behaviour of yeast towards various desiccating agents. 
When yeast is dried at the ordinary temperature it retains its vitality 
for a considerable period. If, however, the dried yeast be heated for 
six hours at 1 00 it loses the power of growth and reproduction but 
still retains that of fermenting sugar, and when ground with sand, 
kieselguhr and 10 per cent, glycerol solution yields an active juice 
[Buchner, 1897, 2 ; 1900, i]. Preparations (known as zymin) obtained 
by treating yeast with a mixture of alcohol and ether [Albert, 1900, 
1901, i], or with acetone and ether [Albert, Buchner, and Rapp, 
1902], show precisely similar properties (p. 38). The proof in this 
case has been carried a step further, for the active juice obtained by 
grinding such acetone-yeast, when precipitated with alcohol and ether, 
yields an amorphous powder, still capable of fermenting sugar. 

The Preparation of Yeast-Juice. 

Buchner's process for the preparation of active yeast-juice is char- 
acterised by extreme simplicity. The yeast employed, which should 
be fresh brewery yeast, is washed two or three times by being sus- 
pended in a large amount of water and allowed to settle in deep 
vessels. It is then collected on a filter cloth, wrapped in a press cloth, 
and submitted to a pressure of about 50 kilos, per sq. cm. for five 
minutes. The resulting friable mass contains about 70 per cent, of 
water and is free from adhering wort. The washed yeast is then 



22 



ALCOHOLIC FERMENTATION 



mixed with an equal weight of silver sand and 0*2 to 0-3 parts of 
kieselguhr, care being taken that this is free from acid. The correct 
amount of kieselguhr to be added can only be ascertained by experi- 
ence, and varies with different samples of yeast. The dry powder 
thus obtained is brought in portions of 300 to 400 grams into a large 
porcelain mortar and ground by hand by means of a porcelain pestle 
fastened to a long iron rod which passes through a ring fixed in the 

wall (Fig. i). The mortar used by 
Buchner has a diameter of 40 cm. and 
the pestle and rod together weigh 8 
kilos. 

As the grinding proceeds the light- 
coloured powder gradually darkens 
and becomes brown, and the mass 
becomes moist and adheres to the 
pestle, until finally, after two to three 
minutes' grinding, it takes the con- 
sistency of dough, at which stage the 
process is stopped. The mass is next 
enveloped in a press cloth and sub- 
mitted to a pressure of 90 kilos, per 
sq. cm. in a hydraulic hand press, the 
pressure being very gradually raised 
in order to avoid rupture of the cloth. 
The cloth required for I ooo grams of 
yeast measures 60 by 75 cm. and is 
previously soaked in water and then 
submitted to a pressure of 50 kilos, 
per sq. cm., retaining about 35 to 40 
c.c. of water. 

The juice runs from the press on 
to a folded filter paper, to remove kieselguhr and yeast cells, and 
passes into a vessel standing in ice water. 

The yield of juice obtained by Buchner in an operation of this 
kind from I kilo, of yeast amounts to 320 to 460 c.c. It may be in- 
creased by re-grinding the press cake and again submitting it to pres- 
sure, and then amounts on the average to 450 to 500 c.c. 

Since the cell membranes constitute about 20 per cent, of the 
weight of the dry yeast, this yield corresponds to more than 60 per 
cent, of the total cell contents of the yeast. It has been computed 
by Will [quoted by Buchner, E, and H., and Hahn, 1903, p. 66] that 




FIG. i. 



ZYMASE AND ITS PROPERTIES 



only about 20 per cent, of the cells are left unaltered by one grinding 
and pressing, and only 4 per cent, after a repetition of the process, 
at least 57 per cent, of the cells being actually ruptured by the double 
process, and the remainder to some extent altered. It seems probable 
from these figures that a certain amount of the juice may be derived 




SPACE oc- 

- CUPIED BY 
xMASS TO BE 

^ PRESSED. 



FIG. 2. 

from the unbroken cells, and Will expressly states that many un- 
broken cells have lost their vacuoles. 

If the yeast be submitted to a process of regeneration, which 
consists in exposure to a well-aerated solution of sugar and mineral 
salts until fermentation is complete, the juice subsequently ob- 



24 ALCOHOLIC FERMENTATION 

tained is more active than that yielded by the original yeast [Albert, 
1899, i]. 

A modified method of grinding yeast was introduced by Mac- 
fadyen, Morris, and Rowland [1900], who placed a mixture of yeast 
and sand in a jacketed and cooled vessel, in which a spindle carrying 
brass flanges was rapidly rotated [Rowland, 1901]. One kilo, of 
yeast ground in this way for 3-5 hours yielded 350 c.c. of juice. 

This grinding process was at first adopted by Harden and Young 
in their experiments but was afterwards abandoned in favour of Buch- 
ner's hand-grinding process, as it was found liable to yield juices of 
low fermenting power, probably on account of inefficient cooling dur- 
ing the grinding process. A slight modification of Buchner's process 
has, however, been introduced, the hand-ground mass being mixed 
with a further quantity of kieselguhr until a nearly dry powder is 
formed, and the mass packed between two layers of chain cloth in 
steel filter plates and pressed out in a hydraulic press at about 2 tons 
to the square inch (300 kilos, per sq. cm.). The press and plates are 
shown in section in Fig. 2. It has also been found convenient to 
remove yeast cells and kieselguhr from the freshly pressed juice by 
centrifugalisation instead of by filtration through paper, and to wash 
the yeast before grinding by means of a filter-press. 

Working with English top yeasts Harden and Young have found 
the yield of juice extremely variable, the general rule being that the 
amount of juice obtainable from freshly skimmed yeast is smaller than 
that yielded by the same yeast after standing for a day or two after 
being skimmed. The yield for 1000 grams of pressed brewer's yeast 
varies from 150 to 375 c - c -> and is on the average about 250 c.c. 

Very fresh yeast occasionally presents the peculiar phenomenon 
that scarcely any juice can be expressed from the ground mass, 
although the latter does not differ in appearance or consistency from a 
mass which gives a good yield. 

Extraction of Zymase from Unground Yeast. 

I. Maceration of Dried Yeast. 

A valuable addition to the methods of obtaining an active solution 
of zymase was made in 1911 by Lebedeff[i9i I, 2 ; 1912, 2 ; see also 
1911, 3, 7, and 1912, i]. This investigator had been in the habit of 
grinding dried yeast with water for preparing samples of yeast-juice 
of uniform character and observed that when the dried yeast was 
digested with sugar solution and the mixture heated, coagulation 



ZYMASE AND ITS PROPERTIES 25 

took place throughout the whole liquid, the proteins of the yeast 
having passed out of the cells. Further examination revealed the 
interesting fact that dried yeast readily yielded an active extract 
when macerated in water for some time. The quality of the resulting 
" maceration extract " depends on a considerable number of factors, 
the chief of which are : (i) the temperature of drying of the yeast ; (2) 
the temperature of maceration ; (3) the duration of maceration ; and (4) 
the nature of the yeast, as well as, of course, the amount of water 
added in maceration. 

In general the yeast should be dried" at 25-3O and then macerated 
with 3 parts of water for 2 hours at 35. 

The temperature of maceration may as a rule be varied, without 
detriment to the product provided that the time of maceration is also 
suitably altered; thus with dried Munich yeast, maceration for 4-5 
hours at 25 is about as effective as 2 hours at 35, whereas treatment 
for a shorter time at 25 or a longer time at 35 produces in general a 
less efficacious extract. Yeast dried at a lower temperature than 25 
tends to yield an extract poor in co-enzyme (p. 59) and hence of low 
fermenting power, this being especially marked at air temperature. 

The subsequent treatment of the yeast during maceration may, 
however, be of great influence in such cases. Thus a yeast dried at 
15 gave by maceration at 25 for 4-5 hours a weak extract (yielding 
with excess of sugar 0*33 g. CO 2 ), whereas when macerated at 35 for 
2 hours it yielded a normal extract (i "36 g. CO 2 ). 

The nature of the yeast is of paramount importance. Thus while 
Munich (bottom) yeast usually gives a good result, a top yeast from a 
Paris brewery was found to yield extracts containing neither zymase 
nor its co-enzyme in whatever way the preparation was conducted. 
The existence of such yeasts is of great interest, and it was probably 
due to the unfortunate selection of such a yeast for his experiments 
that Pasteur was unable to prepare active fermenting extracts and 
therefore failed to anticipate Buchner by more than 30 years (see p. 
I 5). The English top yeasts as a rule give poor results [see Dixon 
and Atkins, 1913] and sometimes yield totally inactive maceration 
extract. It is not understood why the enzyme passes out of the cell 
during the process of maceration and the whole method gives rise to 
a number of extremely interesting problems. 

Method. A suitable yeast is washed by decantation, filtered 
through a cloth, lightly pressed by means of a hand press, and then 
passed through a sieve of 5 mm. mesh, spread out in a layer i-i -5 cm. 
thick and left at 25-35 for two days. Fifty grams of the dried yeast is 



26 ALCOHOLIC FERMENTATION 

thoroughly and carefully mixed with 1 50 c.c. of water in a basin by means 
of a spatula and the whole digested for two hours at 3 5. The mass often 
froths considerably. It is then filtered through ordinary folded filter 
paper, preferably in two portions, and collected in a vessel cooled by 
ice. The separation may also be effected by centrifuging or pressing 
out the mass, and the maceration may be conveniently conducted in 
a flask immersed in the water of a thermostat. It is not advisable to 
macerate more than 50 grams in one operation. Under these con- 
ditions 25-30 c.c. of extract are obtained after 20 minutes' filtration, 
70-80 c.c. in twelve hours. Dried Munich yeast can be bought from 
Messrs. Schroder of Munich and serves as a convenient source of the 
extract. 1 

This extract closely resembles in properties the juice obtained by 
grinding the same yeast, but it is usually more active and contains 
more inorganic phosphate (see p. 46). 

2. Other Methods. 

Attempts to prepare active extracts from undried yeast in an 
analogous manner have so far not been very successful. Thus Rinckle- 
ben [1911] found that plasmolysis by glycerol (8 per cent.) or sodium 
phosphate (5 per cent.) sometimes yielded an active juice and sometimes 
a juice which contained enzyme but no co-enzyme, but more often an 
inactive juice incapable of activation (p. 64) [see also Kayser, 
1911]. 

Giglioli [191 1 ] by the addition of chloroform also obtained an active 
liquid. It appears in fact as though almost any method of plasmoly- 
sing the yeast cell may yield a certain proportion of zymase in the 
exudate. 

An ingenious process has been devised by Dixon and Atkins 
[1913] who applied the method of freezing in liquid air which they 
had found efficacious for obtaining the sap from various plant organs. 
They thus succeeded in obtaining from yeast, derived from Guinness' 
brewery in Dublin, liquids capable of fermenting sugar and of about 
the same efficacy as the maceration extracts prepared by Lebedeff's 
method from the same yeast. The results were, however, in both 
cases very low, the maximum total production of CO 2 by 25 c.c. of liquid 
from excess of sugar being 32*5 c.c. (air temperature) or about 0-06 g. 
Munich yeast on the other hand yields, either by maceration or 
grinding, a liquid giving as much as 1*5-2 g. of CO 2 per 25 c.c., whilst 

1 The material supplied is occasionally found to yield an inactive extract and every 
sample should be tested. 



ZYMASE AND ITS PROPERTIES 27 

English yeast-juice prepared by grinding often gives as much as 0*5- 
07 g. of C0 2 . 

No direct comparison with the juice prepared by grinding was 
made by Dixon and Atkins, but it may be concluded from their results 
that the best method of obtaining an active preparation from the top 
yeasts used in this country is that of grinding. Maceration, freezing 
and plasmolysis alike yield poor results. With Munich yeast on the 
other hand the maceration process yields excellent results, whilst the 
liquid air process has not so far been tried. 

Practical Methods for the Estimation of the Fermenting 
Power of Yeast-Juice. 

In order to estimate the amount of carbon dioxide evolved in a 
given time and the total amount evolved by the action of yeast-juice 
on sugar, Buchner adopted an extremely simple method, which con- 
sisted in carrying out the fermentation in an Erlenmeyer flask pro- 
vided with a small wash-bottle, which contained sulphuric acid and was 
closed by a Bunsen valve, and ascertaining the loss of weight during 
the experiment. Corrections are necessary for the carbon dioxide 
present in the original juice and retained in the liquid at the close of 
the experiment and for that present in the air space of the apparatus, 
but it was found that for most purposes these could be neglected. In 
cases in which greater accuracy was desired, the carbon dioxide was 
displaced by air before the weighings were made. A typical experi- 
ment of this kind, without displacement of carbon dioxide, is the follow- 
ing : 

March 22, 1899, Berlin bottom yeast V. 20 c.c. juice + 8 grams cane sugar + 
0*2 c.c. toluene as antiseptic at 16. Grams of carbon dioxide after 
24 48 72 96 hours. 

0*40 0-64 o'99 I'll 

The total weight of carbon dioxide evolved under these conditions 
is termed the fermenting power of the juice (Buchner). 

A more accurate method [Macfadyen, Morris, and Rowland, 1900] 
consists in passing the carbon dioxide into caustic soda solution 
and estimating it by titration. The yeast-juice, sugar, and antiseptic 
are placed in an Erlenmeyer flask provided with a straight glass tube, 
through which air can be passed over the surface of the liquid, and a 
conducting tube leading into a second flask which contains 50 c.c. of 
i o per cent, caustic soda solution and is connected with the air by a 
guard tube containing soda lime. The juice can be freed from carbon 
dioxide by agitation in a current of air before the flask is connected to 



28 



ALCOHOLIC FERMENTATION 



that containing the caustic soda solution, and at the end of the period 
of incubation air is passed through the apparatus, the liquid being 
boiled out if great accuracy is required. The absorption flask is then 
disconnected and the amount of absorbed carbon dioxide estimated by 
titration. This is carried out by making up the contents of the flask 
to 200 c.c., taking out an aliquot portion, rendering this exactly neutral 
to phenophthalein by the addition first of normal and finally of deci- 
normal acid, adding methyl orange and titrating with decinormal acid 
to exact neutrality. Each c.c. of decinormal acid used in this last titra- 
tion represents 0*0044 gram of carbon dioxide in the quantity of solution 
titrated. 

These methods are only suitable for observations at considerable 
intervals of time. For the continuous observation of the course of fer- 




FIG. 3. 

mentation Harden, Thompson and Young [1910] connect the ferment- 
ation flask with a Schiffs azotometer filled with mercury and measure the 
volume of gas evolved, the liquid having been previously saturated 
with carbon dioxide (Fig. 3). The level of the mercury in the reser- 
voir is kept constant by a syphon overflow, as shown in the figure, or, 
according to a modification introduced by S. G. Paine, by a specially 
constructed bottle provided with two tubulures near the bottom. This 
ensures that no change in the pressure in the flask occurs, and the 
volume of gas observed is reduced to normal pressure by means of a 
table. Before making a reading it is necessary to shake the fermenting 
mixture thoroughly, as the albuminous liquid very readily becomes 
greatly supersaturated with carbon dioxide, so much so in fact that 
very little gas is evolved in the intervals between the shakings. The 
exact procedure in making an observation consists in shaking the flask 



ZYMASE AND ITS PROPERTIES 29 

thoroughly, replacing in the thermostat, allowing to remain for one 
minute, and then reading the level of the mercury in the azotometer. 
After the required time, say five minutes, has elapsed from the time 
at which the flask was first shaken, it is again removed from the bath, 
shaken as before, replaced, allowed to remain for one minute and the 
reading then taken. In this way readings can be conveniently made 
at intervals of three or five minutes or even less, and much more de- 
tailed information obtained about the course of the reaction than is 
possible by means of observations made at intervals of several hours. 

Another form of volumetric apparatus, designed by Walton [1904], 
has been used by LebedefT [1909]. 

An apparatus on a different principle has been designed by Slator 
[1906] for use with living yeast, but is equally applicable to yeast- 
juice, and a very similar form has been more recently employed by 
Iwanoff [1909, 2]. In this apparatus the change of pressure pro- 
duced by the evolution of carbon dioxide is measured at constant 
volume, and comparative rates of evolution can be obtained with con- 
siderable accuracy, although the method has the disadvantage that the 
absolute volume of gas evolved is not measured. The apparatus con- 
sists of a bottle or flask connected with a mercury manometer. The 
fermenting mixture is placed in the bottle along with glass beads to 
facilitate agitation, the pressure is reduced to a small amount by the 
water-pump, and the rise of pressure is then observed at intervals, this 
being proportional to the volume of gas produced. As in the pre- 
ceding case, the liquid must be well shaken before a reading is made. 

The Alcoholic Fermentation of the Sugars by Yeast-Juice. 

Yeast-juice brings about a slow fermentation of those sugars which 
are fermented by the yeast from which it is prepared as well as of 
dextrin, and of starch and glycogen, which are not fermented by living 
yeast. 

(a) Relation to Fermentation by living Yeast. 

Both in rate of fermentation and in the total fermentation produced, 
yeast-juice stands far behind the equivalent amount of living yeast. 
Taking 25 c.c. of yeast-juice to be equivalent to at least 36 grams of 
pressed yeast containing 70 per cent, of moisture, it is found that whereas 
the yeast-juice (from English top yeast) gives with glucose a maximum 
rate of fermentation of about 3 c.c. in five minutes, the living yeast 
ferments the sugar at the rate of about 126 c.c. in the same time, or 



30 ALCOHOLIC FERMENTATION 

about forty times as quickly. The total carbon dioxide obtainable 
from the yeast-juice, moreover, corresponds to the fermentation of only 
2 to 3 grams of sugar, whilst the living yeast will readily ferment a 
much larger quantity, although the exact limit in this respect has not 
been accurately determined. The reasons for this great difference in 
behaviour will be discussed later on, after the various factors concerned 
in fermentation have been considered (p. 123). 

(b) Relation of Alcohol to Carbon Dioxide. 

In all cases of fermentation by yeast-juice and zymin, the relative 
amounts of carbon dioxide and alcohol produced are substantially in 
the ratio of the molecular weights of the compounds, that is as 44 : 46, 
so that for I part of carbon dioxide I -04 of alcohol are formed. This 
has been shown for the juice and zymin from bottom yeasts by Buchner 
[Buchner, E. and H., and Hahn, 1903, pp. 210, 21 1], who obtained the 
ratios 1*01, 0*98, 1*01, and 0^99 from experiments in which from 8 to 
15 grams of alcohol were produced. Similar numbers, 0-90, IT 2, 
o 95, 0*91 and 0*92, have been obtained for the juice from top yeasts by 
Harden and Young [1904], who worked with much smaller quantities. 
The variable results obtained with juice from top yeast by Macfadyen, 
Morris and Rowland [1900], have not been confirmed. 

(c) Relation of Carbon Dioxide and Alcohol Produced to the Amount 

of Sugar Fermented. 

The construction of a balance-sheet between the sugar fermented 
and the products formed is of special interest in the case of alcoholic 
fermentation by yeast-juice, because, there being no cell growth as in 
the case of living yeast, an opportunity appears to be afforded of as- 
certaining whether the whole of the sugar is converted into alcohol 
and carbon dioxide, or whether some fraction of the sugar passes into 
any of the well-known subsidiary products of alcoholic fermentation by 
yeast, such as glycerol, fusel oil, or succinic acid. Unfortunately the 
question cannot be settled in this way. When the loss of sugar 
during the fermentation is estimated directly, it is usually found to be 
considerably greater than the sum of the alcohol and carbon dioxide 
produced from it. This fact was first observed by Macfadyen, Morris and 
Rowland [1900], and was then confirmed by Buchner [Buchner, E. 
and H., and Hahn, 1903, p. 212], in one instance, the excess of sugar 
lost over products being in this case about 1 5 per cent, of the total 
sugar which had disappeared. The matter was then more thoroughly 
investigated by Harden and Young [1904]. 



2YMASE AND ITS PROPERTIES 31 

The conditions under which the experiment must be carried out 
are not very favourable to the attainment of extreme accuracy. Yeast- 
juice contains glycogen and a diastatic enzyme which converts this 
into dextrins and finally into sugar. This process goes on throughout 
fermentation, tending to increase the sugar present and to make the ap- 
parent loss of sugar less than the sum of the products. In spite of this it 
was found that a certain amount of sugar invariably disappeared without 
being accounted for as alcohol or carbon dioxide, and this whether the 
fermentation lasted sixty or a hundred and eight hours, and inde- 
pendently of the dilution of the juice. This disappearing sugar 
amounted in some cases to 44 per cent, of the total loss of sugar, and 
on the average of twenty-five experiments was 38 per cent. Further 
information was sought by converting all the sugar-yielding con- 
stituents of the juice into sugar by hydrolysis before and after the 
fermentation. This process revealed the fact that when the glucose 
equivalent of the juice before and after fermentation was determined 
after hydrolysis with three times normal acid for three hours (and a cor- 
rection made for the loss of reducing power experienced by glucose 
itself when submitted to this treatment), the difference was almost 
exactly equal to the alcohol and carbon dioxide produced. In other 
words, accompanying fermentation, a change proceeds by which sugar 
is converted into a less reducing substance, reconvertible into sugar 
by hydrolysis with acids. Similar results were subsequently obtained 
by Buchner and Meisenheimer [1906], who employed 1-5 normal acid 
and observed a small nett loss of sugar. Still more recently LebedefT 
[1909, 1910, see also 1913, 2] has carried out similar estimations 
with the same result. It is doubtful whether the experiments which 
have so far been made on this point are sufficiently accurate to decide 
with certainty whether or not the loss of sugar is exactly equal to the 
sum of the carbon dioxide and alcohol produced. It has been shown 
by Buchner and Meisenheimer [1906] that glycerol is a constant pro- 
duct of alcoholic fermentation by yeast-juice (p. 95), and no other source 
for this than the sugar has yet been found, so that it is not improbable 
that a small amount of sugar is converted into non-carbohydrate sub- 
stances other than carbon dioxide and alcohol. 

It has also been shown [Harden and Young, 1913] that the deficit 
of sugar is not due to the formation of hexosephosphate (p. 47), 
which has a lower reduction than glucose, and that the solution from 
which the sugar (either glucose or fructose) has disappeared actually 
contains some substance of relatively high dextrorotation and of low 
reducing power. 



32 ALCOHOLIC FERMENTATION 

However this may be, it may be considered as established that 
during alcoholic fermentation sugar is converted by an enzyme into 
some compound of less reducing power, which again yields sugar on 
hydrolysis with acids. The exact nature of this substance has not 
been ascertained, but it appears likely that the process is a synthetical 
one resulting in the formation of some polysaccharide, possibly inter- 
mediate between the hexoses and glycogen. 

A similar phenomenon has been observed with living yeast by 
Euler and Johansson [1912, i], and Euler and Berggren [1912], whose 
interpretation of the observation is discussed later on (p. 57). 

(d) Fermentation of Different Carbohydrates. Autofermentation. 

Yeast-juice and zymin ferment all the sugars which are fermented 
by the yeast from which they are prepared, and, in addition, a number 
of colloidal substances which cannot pass through the membrane of the 
living yeast cell, but which are hydrolysed by enzymes in the juice 
and thus converted into simpler sugars capable of fermentation [Buchner 
and Rapp, 1 898, 3 ; 1 899, 2], Of the simple sugars which have been ex- 
amined, glucose, fructose, and mannose are freely fermented, 1-arabinose 
not at all, whilst the case of galactose is doubtful. Galactose is, however, 
fermented by juice prepared from a yeast which has been " trained " to 
ferment galactose [Harden and Norris, 1910]. As regards both the 
rate of fermentation and the total amount of carbon dioxide evolved 
from glucose and fructose by the action of a definite amount of yeast- 
juice, Buchner and Rapp obtained practically identical numbers. 
Harden and Young [1909], using juice from top yeast, found that 
fructose was slightly more rapidly fermented and gave a somewhat 
larger total than glucose, whilst mannose was initially fermented at 
almost the same rate as glucose, but gave a decidedly lower total, the 
following being the average result : 

Sugar. Relative Rates. Relative Totals. 
Glucose .... i i 

Fructose .... 1-29 1-15 

Mannose .... 1*04 0-67 

Among the disaccharides, cane sugar and maltose are freely fer- 
mented, and the juice can be shown like living yeast to contain inver- 
tase and maltase. The extent of fermentation does not differ materially 
from that attained with glucose. Lactose is not fermented 

Of the higher sugars raffinose is fermented by juice from bottom 
yeast, but more slowly than cane sugar or maltose. No experiments 
seem to have been made with juice from top yeast. 



ZYMASE AND ITS PROPERTIES 33 

As regards the fermentation of the higher carbohydrates, very little 
experimental work has been carried out. Buchner and Rapp found 
that the fermentation of starch paste was doubtful, but that soluble 
starch and commercial dextrin were fermented with some freedom. No 
special study has been made of the diastatic enzymes which bring about 
the hydrolysis of these substances. 

The fermentation of glycogen by yeast-juice is of considerable 
interest, since it is known that the characteristic reserve carbo- 
hydrate of the yeast cell is glycogen [see Harden and Young, 1902, 
where the literature is cited], and moreover that in living yeast the 
intracellular fermentation of glycogen proceeds readily, whereas 
glycogen added to a solution in which yeast is suspended is not 
affected. Yeast-juice contains a diastatic enzyme which hydrolyses 
glycogen to a reducing and fermentable sugar, so that in a juice poor 
in zymase to which glycogen has been added, the amount of sugar is 
found to increase, the hydrolysis of the glycogen proceeding more 
quickly than the fermentation of the resulting sugar [Harden and 
Young, 1904], but the course of this enzymic hydrolysis of glycogen 
by yeast-juice has not yet been studied. As a rule, it is found both 
with juices from top and bottom yeast that the evolution of carbon 
dioxide from glycogen proceeds less rapidly and reaches a lower total 
than from an equivalent amount of glucose. 

Since nearly all samples of yeast contain glycogen, yeast-juice and 
also zymin usually contain this substance as well as the products of its 
hydrolysis. These provide a source of sugar which enters into alco- 
holic fermentation, so that a slow spontaneous production of carbon 
dioxide and alcohol proceeds when yeast-juice is preserved without any 
addition of sugar. The extent of this autofermentation varies consider- 
ably, as might be expected, with the nature of the yeast employed .or 
the preparation of the material, but is generally confined within the 
limits of O'O6 to 0-5 gram of carbon dioxide for 25 c.c. of juice. 

In juice from bottom yeast it amounts to about 5 to 10 per cent, 
of the total fermentation obtainable with glucose [Buchner, 1900, 2], 
whereas in juice from top yeasts, which gives a smaller total ferment- 
ation with glucose, it may occasionally equal, or even exceed, the 
glucose fermentation, and frequently amounts to 30 to 50 per cent of 
it. It is therefore generally advisable in studying the effect of yeast- 
juice on any particular substance to ascertain the extent of auto- 
fermentation by means of a parallel experiment. 

The maceration extract of Lebedeff (p. 24) is usually, but not in- 
variably [Oppenheirner, 1914, 2], free from glycogen, which is hydro- 

3 



34 



ALCOHOLIC FERMENTATION 



lysed and fermented during the processes of drying and macerating, 
and therefore as a rule shows no appreciable autofermentation. 

(e) Effect of Concentration of Sugar on the Total Amount of 
Fermentation. 

The kinetics of fermentation by zymase will be considered later on 
(p. 1 20), but the effect on the total fermentation of different concentra- 
tions of sugar, this substance being present throughout in considerable 
excess, may be advantageously discussed at this stage. The subject 
has been investigated by Buchner [Buchner, E. and H., and Hahn, 1903, 
pp. 150-8; Buchner and Rapp, 1897] using cane sugar, and he has 
found both for yeast-juice and for dried yeast-juice dissolved in water 
that (a) the total amount of fermentation increases with the concentra- 
tion of the sugar ; (ft) the initial rate of fermentation decreases with 
the concentration of the sugar. The following are the results of a 
typical experiment, 20 c.c. of yeast-juice being employed in presence 
of toluene at 22 : 



Cane Sugar. 


COg in grams after 


Weight. 


Per cent. 


6 hours. 


24 hours. 


96 hours. 


2'2 


IO 


0-17 


O'5O 


0'55 


3*52 


15 


0-14 


0'53 


0-64 


5 


20 


0-13 


'54 


073 


6-66 


25 


0-13 


0-52 


0-80 


8-56 


30 


O'I2 


0-46 


0-81 


10-76 


35 


O*I2 


0*40 


0*82 


I3'33 


40 


O'll 


0-36 


0-82 



The results as to the total fermentations in experiments of this 
kind are liable to be vitiated by the circumstance that when a low 
initial concentration of sugar is employed, the supply of sugar may be 
so greatly exhausted before the close of the experiment as to cause a 
marked diminution in the rate of fermentation and hence an unduly 
low total. Even allowing, however, for any effect of this kind, the 
foregoing table clearly shows the increase in total fermentation and 
the decrease in initial rate accompanying the increase of sugar concen- 
tration from 10 to 40 percent. Working with a greater range of 
concentrations (3*3-53*3 grm. per 100 c.c.) Lebedeff has obtained 
similar results with maceration extract [1911, 4], but has found that 
the total amount fermented diminishes after a certain optimum con- 
centration (about 33-3 grm. per 100 c.c.) is reached. 

A practical conclusion from these experiments is that a high 



ZYMASE AND ITS PROPERTIES 



35 



concentration of sugar tends to preserve the enzyme in an active state 
for a longer time. Simultaneously it prevents the development of 
bacteria and yeast cells. 

(/) Effect of Varying Concentration of Yeast-Juice. 

This subject, which is of considerable importance with reference to 
the question of the protoplasmic or enzymic nature of the active agent 
in yeast-juice, has been examined in some detail by Buchner [Buchner, 
E. and H., and Hahn, 1903, pp. 158-65] and by Meisenheimer [1903] 
for juices from bottom yeast, by Harden and Young [1904] for those 
from top yeast, and by Lebedeff [1911, 4] for maceration extract, the 
results obtained being in substantial agreement. 

Dilution of yeast-juice with sugar solution, so that the concentra- 
tion of the sugar remains constant, produces a small progressive 
diminution in the total fermentation, which only becomes marked 
when more than 2 volumes are added, and this independently of the 
actual concentration of the sugar. Dilution with water produces a 
somewhat more decided diminution, which, however, does not exceed 
50 per cent, of the total for the addition of 3 volumes of water. The 
effect on maceration extract is somewhat greater but of the same kind. 
The autofermentation of juice from top yeast is scarcely affected by 
dilution with 4 volumes of water. 



Nature of Juice. 


Per cent, of Sugar 
Employed by Weight. 


Volumes of Sugar 
Solution Added. 


Volumes of 
Water Added. 


Total Fermentation 
in g. of CO 2 . 




I 


2Q 


O 





0-99 








I 


- 


1*13 








2 





O'92 


^ 






4 





079 


5 


2 


9 


O 





0-43 


> 






I 





0*60 


| 






2 





0'53 


i 






4 





0-41 


H 


3 


9 





O 


0*46 











I 


0-32 











2 


0'33 











3 


0-36 




I 





_ 


O 


O'29 






(Autofermentation) 





2 


0*29 











3 


0-28 




2 


29 


O 





0-31 









I 





o-34 


I 






2 





0-31 


DM 

n. 






4 





0'35 




H 






6 





0'28 




3 


7'4 








o'44 











I 


0'35 











2 


0-30 










3 


0-28 



ALCOHOLIC FERMENTATION 



On the whole, therefore, yeast-juice maybe said to be only slightly 
affected by dilution even with pure water, and the effect of the latter 
can in no way be regarded as comparable with the poisonous effect 
which it exerts on living protoplasm, as suggested by Macfadyen, 
Morris, and Rowland [1900]. 

() The Effect of Antiseptics on the Fermentation of Sugars by 

Yeast- Juice. 

Buchner has paid special attention to the effect of antiseptics 
on the course of fermentation by yeast-juice [Buchner and Rapp, 1897 ; 
1898, 2, 3; 1899, i; Buchner and Antoni, 1905, i; Buchner and 
Hoffmann, 1907 ; Buchner, E. and H.,and Hahn, 1903, pp. 169-205 ;see 
also Albert, 1899,2; Gromoffand Grigorieff, 1904; Duchac'ek, 1909] 
in order (i) to obtain evidence as to the possibility of the active agent 
in yeast-juice consisting of fragments of protoplasm and not of a 
soluble enzyme, and (2) also to provide a safe -method of avoiding 
contamination, by the growth of bacteria or yeasts, of the liquids used 
which were often kept at 25 for several days. The results of these 
experiments are briefly summarised in the following table, in which 
the effect of each substance on the total fermentation produced is 
noted : 



Substance. 
Concentrated solution of glycerol 

sugar 

Toluene (to saturation or excess) 
Chloroform 0*5 per cent. 

,, 0*8 per cent, (saturation) 

Large excess (17 per cent. 

Chloral hydrate 0-7 per cent. . 

3-5-5-4 Per cent. 
Phenol O'l per cent. 

0-5 



Thymol 
Benzoic acid 
Salicylic acid 

Formaldehyde 
H 
Acetone 

Alcohol 


i 
5 

O'l 

0-25 

O'l 

0-27 

O'I2 
0-24 

6 
6 



Sodium fluoride 0^5 

2 

Ammonium fluoride 0*55 per cent. 
Sodium azoimide, NaN 3 , 0-36 per cent. 

,i 071 

Quinine hydrochloride i ,, 

Ozone io*4-34'8 milligrams per 20 c.c. 
Hydrocyanic acid 1*2 per cent. 



Effect on Total Fermentation. 
Slight diminution 

,, increase 

Less than 10 per cent, diminution 
Slight increase 
No change 

64 per cent, diminution 
Increase up to 27 per cent. 
Completely destroyed 
No change 

40 per cent, diminution 
Completely destroyed 
Slight diminution 
Marked ,, 
7 per cent, diminution 
26 
10 
35 

20 

30-60 

20 

80 

O-2O 

75 

90 

Almost completely destroyed 

Completely destroyed 

Slight diminution 

Marked 

Slight increase 

Marked diminution 

Completely destroyed 



ZYMASE AND ITS PROPERTIES 37 

The general result of these experiments is to show that quantities 
of antiseptics which are sufficient to inhibit the characteristic action 
of living cells have only a slight effect on the fermentative activity of 
yeast-juice. A large excess of the antiseptic in many cases produces 
a very decided diminution or total destruction of the fermenting 
power, and accompanying this a precipitation of the constituents of the 
juice. The decided increase of activity produced by small quantities 
of chloral hydrate, and to a less marked extent by chloroform and a 
few other substances, is of considerable interest* It is ascribed by 
Duchaek to a selective action on the proteoclastic enzyme, but with- 
out satisfactory evidence. 

Hydrocyanic acid, even in dilute solution, completely suspends 
the fermenting power of the juice, without, however, producing any 
permanent change in the fermenting complex, as is shown by the 
fact that when the hydrocyanic acid is removed by a current of air, 
the juice regains its fermenting power. In this respect hydrocyanic 
acid behaves precisely as with many other enzymes and with colloidal 
platinum [Bredig, 1901]. Sodium arsenite is a pronounced proto- 
plasmic poison, which rapidly destroys the power of growth and repro- 
duction in living cells, and was therefore applied to yeast-juice 
to differentiate between protoplasmic and enzymic action. It was, 
however, found that the action of this substance was complicated by 
some unknown factor and very irregular results were obtained [Buchner, 
E. and H., and Hahn, 1903, pp. 193 ff.]. These phenomena appear 
to be of the same order as those produced by the addition of arsenates 
to yeast-juice [Harden and Young, 1906, 3], and will be discussed 
along with the latter (p. 77). 

Permanent Preparations Containing Active Zymase. 

A considerable number of preparations have been obtained in the 
dry state which retain some proportion of the fermenting power of 
yeast or yeast-juice. 

Starting with yeast-juice, it is possible to arrive at this result either 
by evaporation or precipitation. When the juice is very rapidly 
evaporated to a syrup at 20 to 25 and then further dried at 35, either 
in the air or in a vacuum and finally exposed over sulphuric acid in 
a vacuum desiccator, a dry brittle mass is obtained which is soluble in 
water and retains practically the whole of the fermenting power of the 
juice. The success of the preparation depends on the nature of the 
yeast from which the juice is derived, Berlin yeasts V and S yielding 
much less satisfactory results than Munich yeast. The powder when 



38 ALCOHOLIC FERMENTATION 

thoroughly dry is found to retain its properties almost unimpaired for 
at least a year, and can be heated to 85 for eight hours without under- 
going any serious loss of fermenting power [Buchner and Rapp, 1898, 
4 ; 1901 ; Buchner, E. and H., and Hahn, 1903, pp. 132-9]. 

Active powders can also be obtained by precipitating yeast-juice 
with alcohol, alcohol and ether, or acetone. The preparation is best 
effected by bringing the juice into 10 volumes of acetone, centrifug- 
ing at once and as rapidly as possible, washing, first with acetone and 
then with ether, and finally drying over sulphuric acid. The white 
powder thus obtained is not completely soluble in water but is almost 
entirely dissolved by aqueous glycerol (2-5 to 20 per cent.), forming 
a solution which has practically the same fermenting power as the 
original juice. The precipitation can be repeated without any serious 
loss of fermenting power. Prolonged contact of the precipitate with 
the supernatant liquid, especially when alcohol or alcohol and ether 
are used, causes a rapid loss of the characteristic property [Albert 
and Buchner, 1900, I, 2 ; Buchner, E. and H., and Hahn, 1903, 
pp. 228-246 ; Buchner and Duchac'ek, 1909]. 

Dry preparations capable of fermenting sugar can also be readily 
obtained from yeast without any preliminary rupture of the cells. 
Heat alone (yielding a product known as hefanol) or treatment with 
dehydrating agents may be used for this purpose, and a brief allusion 
has already been made (p. 21) to the different varieties of permanent 
yeast (Dauerhefe) obtainable in these ways. The most important of these 
products are the dried Munich yeast (Lebedeff, see p. 25), and the 
material known as zymin, which is now made under patent rights for 
medicinal purposes by Schroder of Munich. The latter has proved of 
value in the investigation of the production of zymase in the yeast cell 
[Buchner and Spitta, 1902], and of many other problems concerned with 
alcoholic fermentation. In order to prepare it 500 grams of finely 
divided pressed brewer's yeast, containing about 70 per cent, of water, 
are brought into 3 litres of acetone, stirred for ten minutes, and 
filtered and drained at the pump. The mass is then well mixed with 
I litre of acetone for two minutes and again filtered and drained. 
The residue is roughly powdered, well kneaded with 250 c.c. ofether for 
three minutes, filtered, drained, and spread on filter paper or porous 
plates. After standing for an hour in the air it is dried at 45 for 
twenty-four hours. About 150 grams of an almost white powder 
containing only 5 '5 to 6-5 percent of water are obtained. This is quite 
incapable of growth or reproduction but produces a very considerable 
amount of alcoholic fermentation, far greater indeed than a correspond- 



ZYMASE AND ITS PROPERTIES 



39 



ing quantity of yeast-juice. Two grams of the powder corresponding 
to 6 grams of yeast and about 3 '5 to 4 c.c. of yeast-juice, are capable of 
fermenting about 2 grams of sugar, whereas the 4 c.c. of yeast-juice 
would on the average only ferment from one-quarter to one-sixth of 
this amount of sugar. The rate produced by this amount of zymin is 
about one-eighth of that given by the corresponding amount of liv- 
ing yeast [Albert, 1900; Albert, Buchner, and Rapp, 1902]. The 
proteoclastic ferment is still present in zymin, which undergoes auto- 
lysis in presence of water in a similar manner to yeast-juice [Albert, 
1901, 2} 

As already mentioned an active juice can be prepared by grinding 
acetone-yeast with water, sand, and kieselguhr, and this process presents 
the advantage that samples of yeast-juice of approximately constant 
composition can be prepared at intervals from successive portions of a 
uniform supply of acetone-yeast. 

Preparations of acetone-yeast, made from yeast freed from glycogen 
by exposure in a thin layer to the air for three or four hours at 35 to 
45, or eight hours at the ordinary temperature [Buchner and Mitscher- 
lich, 1904], show practically no autofermentation and may be used 
analytically for the estimation of fermentable sugars. 

All the foregoing preparations exhibit the same general properties 
as yeast-juice, as regards their behaviour towards the various sugars, 
antiseptics, etc. 

When zymin is mixed with sugar solution without being previously 
ground, it exhibits a peculiarity which is of some practical interest. 
The time which elapses before the normal rate of fermentation is 
attained and the total fermentation obtainable vary with the amount of 
sugar solution added, the time increasing and the total diminishing as 
the quantity of this increases. This phenomenon appears to have been 
noticed by TrommsdorfT [1902], and a single experiment of Buch- 
ner shows the influence of the same conditions [Buchner, E. and H., and 
Hahn, 1903, p. 265, Nos. 700-1], Harden and Young have found 
that when 2 grams of zymin are mixed with varying quantities of 10 
per cent sugar solution the following results are obtained : 



Volumes of Sugar Solution. 


Total Gas Evolved in 


i hour. 


2 hours. 


3 hours. 


4 hours. 


22-5 hours. 


5c.c 

10 . 

20 . 

40 . 


15*7 
2'2 
O'Q 

1*4 


31-6 
10-5 
2-4 
17 


44-8 

23 
I 3 -6 

2-3 


56-5 
31'8 

237 
2-Q 


2333 
202-3 
125-5 
56-3 





40 ALCOHOLIC FERMENTATION 

This behaviour appears to be due to the removal of soluble matter 
essential for fermentation from the cell, which is discussed later on. It 
follows that when zymin is being tested for fermenting power, a uni- 
form method should be adopted, and all comparative tests should be 
made with the same volumes of added sugar solution. Ground zymin 
appears to begin to ferment somewhat more slowly than unground 
(2 grm. to 1 2 -4 c.c. of sugar solution in each case), but eventually 
produces the same total volume of gas [Buchner and Antoni, 1905, i]. 



CHAPTER III. 

THE FUNCTION OF PHOSPHATES IN ALCOHOLIC FERMENTATION. 

IN the course of some preliminary experiments (commenced by the 
late Allan Macfadyen, but subsequently abandoned) on the production 
of anti-ferments by the injection of yeast-juice into animals, the 
serum of the treated animals was tested for the presence of such anti- 
bodies both for the alcoholic and proteoclastic enzymes of yeast-juice, 
and it was then observed that the serum of normal and of treated 
animals alike greatly diminished the autolysis of yeast-juice. 

As the explanation of the comparatively rapid disappearance of 
the fermenting power from yeast-juice had been sought, as already 
mentioned (p. 20), in the hydrolytic action of the tryptic enzyme 
which always accompanies zymase, the experiment was made of 
carrying out the fermentation in the presence of serum, with the result 
that about 60 to 80 per cent, more sugar was fermented than in the 
absence of the serum [Harden, 1903]. 

This fact was the starting-point of a series of attempts to obtain a 
similar effect by different means, in the course of which a boiled and 
filtered solution of autolysed yeast-juice was used, in the hope that the 
products formed by the action of the tryptic enzyme on the proteins 
of the juice would, in accordance with the general rule, prove to be an 
effective inhibitant of that enzyme. This solution was, in fact, found 
to produce a very marked increase in the total fermentation effected 
by yeast-juice, the addition of a volume of boiled juice equal to that 
of the yeast-juice doubling the amount of carbon dioxide evolved 
[Harden and Young, 1905, i]. This effect was found to be common 
to the filtrates from boiled fresh yeast-juice and from boiled autolysed 
yeast-juice, and was ultimately traced in the main, not to the anti- 
try ptic effect which had been surmised, but to two independent factors, 
either of which was capable in some degree of bringing about the 
observed result. 

Boiled yeast-juice was indeed found to possess a decided anti- 
autolytic effect, as determined by a comparison of the amounts of 
nitrogen rendered non-precipitable by tannic acid in yeast-juice alone 

41 



42 ALCOHOLIC FERMENTATION 

and in a mixture of yeast-juice and boiled juice on preservation 
[Harden, 1905]. The anti-autolytic effect, however, appeared to 
vary independently of the effect on the fermentation, and the con- 
clusion was drawn, as stated above, that the increase in the alcoholic 
fermentation was not directly dependent on the decrease in the action 
of the proteoclastic enzyme but was due to some independent cause. 
The property possessed by boiled yeast-juice of diminishing the auto- 
lysis of yeast-juice has now been carefully examined by Buchner and 
Haehn [1910, 2] and ascribed by them to a soluble anti-protease 

(p. 65). 

The two factors to which the increase in fermentation produced by 
the addition of boiled juice were ultimately traced were (i) the pre- 
sence of phosphates in the liquid, and (2) the existence in boiled fresh 
yeast-juice of a co-ferment or co-enzyme, the presence of which is 
indispensable for fermentation [Harden and Young, 1905, i, 2]. 

The former of these factors will be here discussed and the co-enzyme 
will form the subject of the following chapter. 

The general fact that sodium phosphate increases the total ferment- 
ation produced by a given volume of yeast juice was observed on 
several occasions by Wroblewski [1901] and also by Buchner [Buchner, 
E. and H., and Hahn, 1903, pp. 141-2], who ascribed the action of this 
salt to its alkalinity, comparing it in this respect with potassium carbonate 
and remarking that the increase in both cases took place chiefly in the 
first twenty hours of fermentation. The increased amount of ferment- 
ation following the addition of boiled yeast-juice was also noted by 
Buchner and Rapp [1899, 2, No. 265, p. 2093] in a single experi- 
ment. 

Observations made at intervals of a few minutes instead of twenty 
hours have, however, revealed the fact that phosphates play a part of 
fundamental importance in alcoholic fermentation and that their pre- 
sence is absolutely essential for the production of the phenomenon. 

Effect of the Addition of Phosphate to a Fermenting Mixture 
of Yeast- Juice and Sugar. 

When a suitable quantity l of a soluble phosphate is added to a 
fermenting mixture of glucose, fructose, or mannose with yeast-juice, 
the rate of fermentation rapidly rises, sometimes increasing as much 
as twenty-fold, continues at this high value for a certain period and 
then falls again to a value approximately equal to, but generally 

1 The effect of an excess of phosphate is discussed later on, p. 71. 



THE FUNCTION OF PHOSPHATES 43 

somewhat higher than, that which it originally had. Careful experi- 
ments have shown that during this period of enhanced fermentation 
the amounts of carbon dioxide and alcohol produced exceed those 
which would have been formed in the absence of added phosphate by 
a quantity exactly equivalent to the phosphate added in the ratio CO 2 
or C 2 H 6 O : R' 2 HPO 4 [Harden and Young, 1906, i]. 

This result is of fundamental importance, and the evidence on 
which it rests deserves some consideration. Quantitative experiments 
on this subject require certain preliminary precautions. The acid 
phosphates are too acid to permit of any extended fermentation and 
the phosphates of the formula R / 2 HPO 4 absorb a considerable volume 
of carbon dioxide with production of a bicarbonate, according to the 

reaction : 

R 2 H PO 4 + H 2 C O 3 =^ RH CO 3 + RH 2 PO 4 . 

The method which has been adopted, therefore, is to employ either 
a secondary phosphate saturated with carbon dioxide at the temper- 
ature of the experiment, or a mixture of five molecular proportions of 
the secondary phosphate with one molecular proportion of a primary 
phosphate, in which the amount of bicarbonate formed is negligible. 
In the former case it is necessary to ascertain whether any of the 
carbon dioxide evolved is derived from the bicarbonate by the action 
of acid originally present or produced in the yeast-juice or by a dis- 
turbance of the original equilibrium owing to the chemical change 
which occurs. This is done by acidifying duplicate samples with 
hydrochloric acid before and after the fermentation and measuring the 
gas evolved in each case. Any necessary correction can then be made. 
The calculation of the extra amount of carbon dioxide evolved from 
yeast-juice containing sugar when a phosphate is added involves an 
estimation of the amount which would have been evolved in the 
absence of added phosphate, and this is a matter of some difficulty. 
Since the final steady rate of fermentation attained is often slightly 
different from the initial rate, the practice has been adopted of ascer- 
taining this final rate and then calculating the total evolution corres- 
ponding to it for the whole period from the time of the addition of 
the phosphate to the end of the observations. This amount deducted 
from the observed total leaves the extra amount of carbon dioxide 
formed, and it is this quantity which is equivalent to the phosphate 
added. Alcohol is simultaneously produced in the normal ratio. 
The justification for this method of calculation will be found later 

(P- 54). 

The following table, containing the results of experiments with 



44 



ALCOHOLIC FERMENTATION 



glucose, fructose, and mannose, indicates very clearly the nature of the 
method of calculation and also of the agreement between observation 
and theory. 

Three quantities of 25 c.c. of yeast-juice + 5 c.c. of a solution con- 
taining i gram of the sugar to be examined (a large excess) were in- 
cubated with toluene at 25 for one hour, in order to remove all free 
phosphate, and to each were then added 5 c.c. of a solution of sodium 
phosphate corresponding to 0-1632 gram of Mg 2 P 2 O 7 and equivalent 
to 32*6 c.c. of carbon dioxide at N.T.P. The rates of fermentation were 
then observed until they had passed through the period of acceleration 
and had fallen and attained a steady value, the gases being measured 
moist at 19*3 and 760*15 mm. 





Glucose. 


Mannose. 


Fructose. 


Maximum rate attained, c.cs. per five minutes . 
Final rate of fermentation ..... 
Total carbon dioxide produced by fermentation in 
fifty-five minutes after addition of phosphate . 
Correction for evolution in absence of phosphate in 


9-6 
1*1 

497 

12*1 


7 
0*96 

47-8 
io'6 


11-3 
i -08 

47-6 

II'Q 


Extra carbon dioxide equivalent to phosphate . 
at N.T.P. 


37'6 

34*4 


37'2 
34 


357 
32-6 



These numbers agree well with the value calculated from the phos- 
phate added, viz. 32*6 [Harden and Young, 1909]. 

Another experiment is illustrated graphically in Fig. 4, in which 
the volume of carbon dioxide evolved is plotted against time. The 
determination was in this case made by adding 25 c.c. of an aqueous 
solution containing 5 grams of glucose to one quantity of 25 c.c. of 
yeast-juice (curve A) and 5 c.c. of o'3 molar solution of the mixed prim- 
ary and secondary sodium phosphates, and 20 c.c. of a solution containing 
5 grams of glucose to a second equal quantity of yeast-juice (curve B). 
Curve A shows the normal course of fermentation of yeast-juice with 
glucose. There is a slight preliminary acceleration during the first 
twenty minutes, due to free phosphate in the juice, and the rate then 
becomes steady at about I -4 c.c. in five minutes. During this prelimin- 
ary acceleration 10 c.c. of extra carbon dioxide are evolved, this number 
being obtained graphically by continuing the line of steady rate back 
to the axis of zero time. Curve B shows the effect of the added phos- 
phate. The rate rises to about 9-5 c.c. in five minutes, i.e. to more 
than six times the normal rate, and then gradually falls until after an 
hour it is again steady and almost exactly equal to 1*4 c.c. per five 
minutes. Continuing the line of steady rate back to the axis of zero 



THE FUNCTION OF PHOSPHATES 



45 



time it is found that the extra amount of carbon dioxide is 48 c.c. 
Subtracting from this the 10 c.c. shown in curve A as due to the juice 
alone, a difference of 38 c.c. is obtained due to the added phosphate. 
The amount calculated from the phosphate added in this case is, at 
atmospheric temperature and pressure, 38-9 c.c. 

After the expiration of seventy minutes from the commencement 
of the experiment, a second addition is made of an equal amount of 
phosphate. The whole phenomenon then recurs, as shown in curve C, 



130 




























































7^^"' 


5^^ 


Hi 


110 
100 
90 
80 

co 60 
3 50 

^40 
|30 

2 10 






















__ ^. 


*-* 


~? 


X 






















~ " ** 






/ 


x c 




























y 


y 




























j 


r 
























































^ 


^ 






















^ . 


**" 


/ 


^ 




























j 


/B 






























/ 












^<*^ 


^-^ 1 
/\ 


** 
















/ 




^^ 


*** 


r*~ 




















^ . 


^ 


^ 


^-ft 


























jL 
































10 ?0 30 40 50 60 70 60 90 100 HO 120 130 140 150 l& 
FIG. 4. 



the maximum rate being slightly lower than before, about 6 c.c. per five 
minutes, and the rate again becoming finally steady at I -4 c.c. as before. 
The extra amount of carbon dioxide evolved in this second period 
obtained graphically as in the former case, is 107 - 68 = 39 c.c. 

It may be noted that in this case the observations after each ad- 
dition last fifty to seventy minutes, so that an error of O'l c.c. per five 
minutes in the estimated final rate would make an error of I to 1*4 c.c. 
in the extra amount of carbon dioxide, i.e. about 3 to 4 per cent, of 
the total, and this is approximately the limit of accuracy of the method. 



46 ALCOHOLIC FERMENTATION 

The results are more precise when the yeast-juice employed is an active 
one, since, when the fermenting power of the juice is low, the initial 
period of accelerated fermentation is unduly prolonged and the cal- 
culation of the extra amount of carbon dioxide is rendered uncertain. 
Zymin (p. 38) yields precisely similar results to yeast-juice, but in 
this case the rate of fermentation is not so largely increased. This has 
the effect that the extra amount of carbon dioxide cannot be quite so 
accurately estimated for zymin, because a slight error in the determin- 
ation of the final rate of fermentation has a greater influence on the result. 
The equivalence between the extra amount of carbon dioxide evolved 
and the phosphate added is, however, unmistakable, as is shown by the 
following results of an experiment with zymin, in which 6 grams of 
zymin (Schroder) + 3 grams of fructose (Schering) + 25 c.c. of water 
were incubated at 25 in presence of toluene until a steady rate had been 
attained. Five c.c. of a solution of sodium phosphate equivalent to 
32*2 c.c. carbon dioxide at N.T.P. were then added. 

Maximum rate attained, c.c. per five minutes 14-1 

Final rate of fermentation 6*2 

Total evolved by fermentation in eighty minutes after addition of phosphate 131 
Correction for evolution in absence of phosphate in eighty minutes . . 99-2 

Extra carbon dioxide at 16 and 767'! mm 31-8 

N.T.P 29-8 

Considering the small proportional rise in rate and the long period of 
accelerated fermentation, the agreement between the volume observed, 
29-8 c.c., and that calculated from the phosphate, 32-2, is quite satis- 
factory [Harden and Young, 1910, i.] Precisely the same relations 
hold for maceration extract, but in this case it must be remembered that 
a large amount of free phosphate is present in the extract, as 
much as 0*3129 grm. Mg 2 P 2 O 7 being obtained from 20 c.c. in one pre- 
paration, so that the original extract had the concentration of a 0-14 
molar solution of sodium phosphate. It is in fact not improbable 
that the delay in the onset of fermentation sometimes observed with 
maceration extract (see Lebedeff, 1912, 2; Neuberg and Rosenthal, 
1 9 1 3 ) may be due to the presence of phosphate in so great an excess of the 
amount which can be rapidly esterified by the enzymes that the rate of 
fermentation is at first greatly lowered (see p. 7 1 ). When this phosphate 
is removed by incubation with glucose or fructose, the subsequent ad- 
dition of phosphate produces the characteristic action and the extra 
carbon dioxide evolved is, as with other yeast preparations, equivalent 
to the phosphate added. An actual estimation carried out in this way 
gave 35 c.c. of CO 2 for an addition of phosphate equivalent to 32-9 
c.c. [Harden and Young, 1912], 



THE FUNCTION OF PHOSPHATES 47 

Within the limits imposed by the experimental conditions, then, the 
fact is well established that the addition of a soluble phosphate to a 
fermenting mixture of a hexose with yeast-juice, maceration extract, 
dried yeast, or zymin causes the production of an equivalent amount 
of carbon dioxide and alcohol. 

This fact indicates that a definite chemical reaction occurs in which 
sugar and phosphate are concerned, and this conclusion is confirmed 
when the fate of the added phosphate is investigated. If an experi- 
ment, such as one of those described above, be interrupted as soon as the 
rate of fermentation has again become normal, and the liquid be boiled 
and filtered, it is found that nearly the whole of the phosphorus present 
passes into the filtrate, but that only a small proportion of this exists 
as mineral phosphate, whilst the remainder, including that added in 
the form of a soluble phosphate, is no longer precipitable by magnesium 
citrate mixture [Harden and Young, 1905, 2]. 

A similar observation was made at a later date by Iwanoff [1907], 
who had previously observed [1905] that living yeast, like many 
other vegetable organisms, converted mineral phosphates into organic 
derivatives. Iwanoff employed zymin and hefanol (p. 38) instead of 
yeast-juice, and found that phosphates were thereby rendered non- 
precipitable by uranium acetate solution, but did not observe the 
accelerated fermentation caused by their addition. 

The foregoing conclusions have been strikingly confirmed by ex- 
periments with maceration extract carried out by Euler and Johansson 
[1913], in which both the carbon dioxide evolved and the phosphate 
rendered non-precipitable by magnesia were determined at intervals. 
When dried yeast is employed as the fermenting agent, the amount 
of phosphate esterified in the earlier stages is greater than would be 
expected, but ultimately becomes exactly equivalent to the carbon 
dioxide evolved. 

Nature of the Phospho-organic Compound formed by Yeast- 
Juice and Zymin from the Hexoses and Phosphate. 

The formation and properties of the compound produced from phos- 
phates in the majiner just described have been investigated by Harden 
and Young [1905, 2; 1908, I ; 1909; 1911, 2], Young [1909; 1911], 
Iwanoff [1907; 1909, i], Lebedeff [1909 ; 1910; 1911, 5,6; 1912, 
3 ; I9i3i i]j and Euler [1912, I ; Euler and Fodor, 1911 ; Euler and 
Kullberg, 1911, 3 ; Euler and Ohlsen, 1 9 1 1 ; 1 9 1 2 ; Euler and Johansson, 
1912, 4; Euler and Backstrom, 1912], but its exact constitution can- 
not as yet be regarded as definitely known. 



48 ALCOHOLIC FERMENTATION 

Phosphates undergo this characteristic change when the sugar 
undergoing fermentation is glucose, mannose, or fructose, and it may 
be said at once that no distinction can be established between the 
products formed from these various hexoses ; they all appear to be 
identical. The compound produced is, as already mentioned, not pre- 
cipitated by ammoniacal magnesium citrate mixture, nor by uranium 
acetate solution. It can, however, be precipitated by copper acetate 
(Iwanoff) and by lead acetate (Young). The preparation of the pure 
lead salt from the liquid obtained by fermenting a sugar with yeast- 
juice or zymin in presence of phosphate is commenced by boiling and 
filtering the liquid. Magnesium nitrate solution and a small quantity 
of caustic soda solution are then added to precipitate any free phos- 
phate, and the liquid well stirred and allowed to stand over night. To 
the neutralised filtrate lead acetate is then added together with sufficient 
caustic soda solution to maintain the reaction neutral to litmus, until 
no further precipitate is formed. The liquid is then filtered or, better, 
centrifugalised, and the precipitate repeatedly washed with water until 
a portion of the clear filtrate gives no reduction when boiled with Fehl- 
ing's solution. It is essential that this washing should be thorough as 
evidence has recently been obtained of the formation under certain con- 
ditions of a hexosephosphate, the lead salt of which is not so sparingly 
soluble as that of the hexosediphosphate [Harden and Robison, 1914]. 
The lead precipitate is then suspended in water, decomposed by a cur- 
rent of sulphuretted hydrogen, the clear filtrate freed from sulphuretted 
hydrogen by a current of air, and finally neutralised with caustic soda. 
The removal of phosphate and conversion into lead salt are repeated 
twice, and the resulting lead salt is then found to be free from nitrogen 
and to have a composition represented by the formula C 6 H 10 O 4 (PO 4 Pb) 2 . 
Lebedeff carries out the preparation in a somewhat different man- 
ner. The fermentation is effected by means of air-dried yeast ( 1 50 grams 
to I litre of water, 210 grams cane-sugar and 105 grams of a mixture of 2 
parts Na 2 HPO 4 and I part NaH 2 PO 4 ) and the liquid (about 700 c.c.) 
after boiling and filtering, is treated with an equal volume of acetone. 
About 300 c.c. of a thick liquid is precipitated and this is redissolved in 
water and precipitated by an equal volume of acetone two or three times. 
The final liquid is then precipitated with warm lead acetate solution 
and filtered and washed with dilute lead acetate solution until the fil- 
trate is clear and no longer reduces Fehling's solution after removal 
of the lead [1910]. Euler and Fodor [1911] on the other hand 
precipitate the free phosphate with magnesia mixture and then add 
acetone, dissolve the syrup thus precipitated in water and add copper 



THE FUNCTION OF PHOSPHATES 49 

acetate solution. A blue copper salt is precipitated which is thoroughly 
washed with water and used for the preparation of solutions of the 
acid. A solution of the free acid can readily be prepared by the action 
of sulphuretted hydrogen on the lead salt suspended in water. It 
forms a strongly acid liquid, which requires exactly two equivalents 
of base for each atom of phosphorus present to render it neutral to 
phenolphthalein. It decomposes when evaporated, leaving a charred 
mass containing free phosphoric acid. The acid is slightly optically 
active, and has [a D ] = + 3 -4. A number of amorphous salts have been 
prepared by precipitation from a solution of the sodium salt, and of these 
the silver, barium, and calcium salts have been analysed with results 
agreeing with the general formula C 6 H 10 O 4 (PO 4 R' 2 ) 2 . The magnesium, 
calcium, barium, and manganese salts, which are only sparingly soluble, 
are all precipitated when their solutions are boiled but re-dissolve on 
cooling, and this property can be utilised for their purification. The 
alkali salts have only been obtained as viscid residues. 

A difference of opinion exists as to the molecular weight and 
constitution of this substance. Iwanoff [1909, i] regards it as a 
triosephosphoric acid, C 3 H 5 O 2 (PO 4 H 2 ), basing this view on the pre- 
paration of an osazone which melted at 142, but when recrystallised 
from benzene gave a product melting at i27-8, which had the same 
appearance, melting-point, and nitrogen content as the triosazone 
formed by the action of phenylhydrazine on the oxidation products of 
glycerol. Neither LebedefT [1909] nor Young could obtain Iwanoffs 
osazone, and all attempts to reduce the acid with formation of 
glycerol either by sodium amalgam or hydriodic acid were unsuccessful 
(Young). There is therefore practically no serious experimental 
evidence in favour of Iwanoff' s view. 

On the other hand, Harden and Young regard the acid as a diphos- 
phoric ester of a hexose. This view is based on the fact that when the 
acid is boiled with water, or an acid, free phosphoric acid is produced 
along with a levo-rotatory solution containing fructose and possibly a 
small proportion of some other sugar or sugars. (Euler and Fodor 
however did not obtain a hexose in this way [1911].) The acid itself 
only reduces Fehling's solution after some hours in the cold, rapidly 
when boiled, whereas when its solution is first boiled, and then treated 
with Fehling's solution in the cold, the products of decomposition bring 
about reduction in a few minutes. The reduction brought about when 
the acid is boiled with Fehling's solution is considerably less (33 per cent.) 
than that produced by an equivalent amount of glucose. The behaviour 
of the compound towards phenylhydrazine is also in complete agree- 

4 - ' 



50 ALCOHOLIC FERMENTATION 

ment with this view. Lebedeff found [1909, 1910] that the acid or its 
salts heated with phenylhydrazine in presence of acetic acid gave an 
insoluble compound which was ultimately found to be the phenyl- 
hydrazine salt of hexosemo nophosphoric acidosazone 

C 6 H 5 NH-NH 2 -H 2 PO 4 -C 4 H 5 (OH) 3 -C(N 2 HC 6 H 6 )CH(N 2 HC 6 H 6 ) 

[Lebedeff, 1910; 1911, 6; Young, 1911]. After recrystallisation 
from alcohol this compound forms yellow needles, melting at 1 51- 15 2. 
It is decomposed by caustic soda yielding a sodium salt 
Na 2 P0 4 -C 4 H 6 (OH) 3 -(CN 2 HC 6 H 5 )-CH(N 2 HC 6 H 5 ) 

and on boiling with caustic soda decomposes giving a hexosazone (free 
from phosphorus) which is probably glucosazone, and in addition 
glyoxalosazone, probably as the result of a secondary decomposition. 
Towards acids it is remarkably stable yielding with hydrochloric acid a 
hexosonephosphoric ester from which the original osazone can be re- 
generated (Lebedeff). Lebedeff at first [1910] argued from the forma- 
tion of this osazone that the original hexosephosphate contained only 
one phosphoric acid group per molecule of hexose. It was however 
shown by Young [1911] and subsequently confirmed by Lebedeff 
[1911, 6] that one molecule of phosphoric acid is split off during the 
formation of the osazone, even in neutral solution. Moreover it has 
been found that in the cold hexosediphosphoric acid reacts with 3 
molecules of phenylhydrazine forming the diphenylhydrazine salt of 
hexosediphosphoric acid phenylhydrazone 

(C 6 H 6 NH-NH 3 -H 2 P0 4 ) 2 -C 6 H 7 (OH) 3 -N 2 HC 6 H 6 . 

This compound crystallises out when I volume of alcohol is added 
to a solution of 3 molecules of phenylhydrazine in one of the acid and 
forms colourless needles melting at 1 1 5-! 1 7. p-Bromophenylhydrazine 
yields an analogous compound melting at I27-I28. 

Precisely the same products are given with phenylhydrazine by the 
hexosephosphoric acid prepared from glucose, mannose, and fructose, 
proving that all these sugars yield the same hexosediphosphoric acid, 
a point of fundamental importance. 

Direct measurements of the molecular weight of the acid by the 
freezing-point method, combined with the determination of the degree 
of dissociation by the rate of cane-sugar inversion, are indecisive, but 
indicate that the acid has a molecular weight considerably higher than 
that required for a triosephosphoric acid. 

A similar uncertainty attaches to the determination of the molecular 
weight from the freezing-point depression and conductivity of the acid 
potassium salt [Euler and Fodor, 1911]. Euler however concludes 



THE FUNCTION OF PHOSPHATES 51 

that both a hexosediphosphoric acid and a triosemonophosphoric acid 
are formed, but has not prepared any derivatives of the latter. 

As regards the constitution of the hexosephosphoric ester several 
suggestions have been made by Young, but no decisive evidence at 
present exists. The identity of the products from glucose, mannose, 
and fructose may be explained by regarding the acid as a derivative 
of the enolic form common to these three sugars (p. 97), or by suppos- 
ing that portions of two sugar molecules may be concerned in its 
production. The formation and composition of the hydrazone and 
osazone are of great importance as they indicate that in all probability 
one of the phosphoric acid residues is united with the carbon atom 
adjacent to the carbonyl group of the hexose. They moreover render 
it certain that the original phosphoric ester is a hexosediphosphoric 
ester and not a triosemonophosphoric ester. 

Hexosediphosphoric acid has not as yet been discovered in the 
animal body. The action of a number of enzymes upon it has been 
examined [Euler, 1912, 2 ; Euler and Funke, 1912; Harding, 1912; 
Plimmer, 1913] with the following results. 

The lipase of castor oil seeds, a glycerol extract of the intestinal 
mucous membrane of the rabbit and pig, and an aqueous extract 
of bran have a slow hydrolytic action, whereas pepsin and trypsin 
are without effect. Feeding experiments with rabbits and dogs 
indicate that the ester is capable of hydrolysis in the animal body, 
a large proportion of the phosphorus being excreted as inorganic 
phosphate. The ester is also decomposed by Bacillus coli communis. 

It is remarkable that the hexosephosphate is not fermented nor 
hydrolysed by living yeast, a fact observed by Iwanoff, Harden and 
Young, and Euler, although, according to the experiments of Paine 
[1911], the yeast cell is at all events partially permeable to the sodium 
salt. 



The Equation of Alcoholic Fermentation. 

An equation can readily be constructed for the reaction in which 
hexosephosphate is formed, the data available being the formula of the 
product and the relation between the phosphate added and the 
carbon dioxide and alcohol produced : 

(i) 2C 6 H 12 6 + 2 P0 4 HR 2 = 2 C0 2 + 2C 2 H 6 + 2 H 2 O + C 6 H 10 O 4 (PO 4 R a ) 3 . 

According to this, two molecules of sugar are concerned in the 
change, the carbon dioxide and alcohol being equal in weight to one 

4* 



52 ALCOHOLIC FERMENTATION 

half of the sugar used, and the hexosephosphate and water representing 
the other half. 

Additional confirmation of this equation is afforded by the deter- 
mination of the ratio between sugar used and carbon dioxide evolved 
when a known weight of sugar together with an excess of phosphate 
is added to yeast-juice at 25. The phenomena then observed are 
precisely similar to those which occur when a phosphate is added to 
a fermenting mixture of yeast-juice and excess of sugar as described 
above. The rate of fermentation rapidly rises and then gradually 
falls until a rate is attained approximately equal to that of the auto- 
fermentation of the juice in presence of phosphate. At this point it is 
found that the extra amount of carbon dioxide evolved, beyond that 
which would have been given off in the absence of added sugar, bears 
the ratio expressed in equation (i) to the sugar added [Harden and 
Young, 1910, 2], The results of four estimations made in this way 
were (a) O'2 grams of glucose gave 26-5 and 2/'9 c.c. of carbon dioxide 
at N.T.P. ; () O'2 grams of fructose gave 27*9 and 28-9 c.c. The 
carbon dioxide calculated from the sugar added in each of the four 
cases is 26^96 c.c. 

It has also been shown by Euler and Johansson [1913] that in 
the fermentation of a mixture of equivalent amounts of phosphate and 
glucose, the whole of the glucose had disappeared when the whole of 
the phosphate had become esterified. 

Cycle of Changes Undergone by Phosphate in Alcoholic 
Fermentation. 

According to equation (i) the free phosphate present .is used up 
in the reaction, and the question at once arises whether there is any 
source from which a constant supply of free phosphate can be elaborated 
in the juice, or whether some other change occurs which results in the 
formation of carbon dioxide and alcohol in the absence of free phos- 
phate. The experimental evidence points in the direction of the 
former of these alternatives, but the question is a very difficult one to 
decide with absolute certainty. 

When a mixture of a phosphate with yeast-juice and sugar is 
examined at intervals and the amount of free phosphate estimated, 
the following stages are observed : 

1. During the initial period of accelerated fermentation following 
the addition of the phosphate, the concentration of free phosphate 
rapidly diminishes. 

2. At the close of this period, the amount of free phosphate 



THE FUNCTION OF PHOSPHATES 53 

present is very low, and, as long as active fermentation continues, no 
marked increase in it occurs. 

3. As alcoholic fermentation slackens and finally ceases, a marked 
and rapid rise in the amount of free phosphate occurs at the expense 
of the hexosephosphate, which steadily diminishes in amount, and 
this change is brought about by an enzyme in the juice and ceases if 
the liquid be boiled. 

This last reaction may be represented by the equation 

(2) C 6 H 10 4 (P0 4 R 2 ) 2 + 2 H 2 = C 6 H 12 6 + aPO^R,. 

In the light of this equation, together with equation No. I, given above, 
all these facts can be simply and easily understood. 

The rapid diminution in the amount of free phosphate during 
stage I corresponds with the occurrence of reaction (i). During the 
whole period of fermentation the enzymic hydrolysis of the hexose- 
phosphate is proceeding according to equation (2). Up to the end of 
stage 2 the phosphate thus produced enters into reaction, according 
to equation (i), with the sugar which is present in excess and is thus 
reconverted into hexosephosphate, so that as long as alcoholic fer- 
mentation is proceeding freely, no accumulation of free phosphate can 
occur. 

As soon as alcoholic fermentation ceases, however, it is no longer 
possible for the phosphate to pass back into hexosephosphate, and 
hence it accumulates in the free state. 

A similar hydrolysis of hexosephosphate and accumulation of 
phosphate occur when a solution of hexosephosphate is treated with 
yeast-juice which has been deprived of the power of fermentation by 
dialysis, or with zymin freed from co-enzyme by washing (p. 63). 

The actual rate of fermentation observed in any particular case in 
presence of excess of sugar, enzyme, and co-enzyme must on this view 
depend on the supply of phosphate which is available. 

In presence of an adequate amount of phosphate, as well as of 
sugar, the highest rate attained represents the maximum velocity at 
which reaction (i) can proceed in that sample of yeast-juice or zymin, 
and this high rate is characteristic of the initial period of accelerated 
fermentation which follows the addition of a suitable quantity of phos- 
phate. By the simple expedient of renewing the supply of phosphate 
as rapidly as it is converted into hexosephosphate, this high rate can 
be maintained for a considerable time [Harden and Young, 1908, 
i]. In this way, for example, an average rate of evolution of carbon 
dioxide of 15 c.c. in five minutes was maintained for an hour and a 







54 ALCOHOLIC FERMENTATION 

quarter, whereas the normal rate in the absence of added phosphate 
was 3 c.c. 

As soon as all the free phosphate has entered into the reaction, 
however, the supply of phosphate depends in the main on the rate at 
which the resulting hexosephosphate is decomposed, and the rate of 
fermentation now attained is conditioned by the rate at which re- 
action (2) proceeds, and this evidently depends on the existing con- 
centration of the hydrolytic enzyme, which may be provisionally termed 
hexosephosphatase. 

The rates attained during the initial period of rapid fermentation 
and the subsequent period of slow fermentation are thus seen to 
represent the velocities of two entirely different chemical reactions. 

These considerations also explain why it is the extra carbon 
dioxide evolved during the initial period, and not the total carbon 
dioxide, which is equivalent to the added phosphate. As the pro- 
duction of phosphate is proceeding throughout the whole period at 
a rate which is equivalent to the normal rate of fermentation, it is 
obviously necessary to deduct the corresponding amount of carbon 
dioxide from the total evolved in order to ascertain the amount equi- 
valent to the added phosphate. 

An explanation is also afforded of the fact that a considerable 
increase in the concentration of hexosephosphate does not materially 
increase the normal rate of fermentation. This is probably due to the 
circumstance that, in accordance with the general behaviour of enzymes 
in presence of excess of the fermentable substance, the hexosephos- 
phatase hydrolyses approximately equal amounts of hexosephosphate 
in equal times whatever the concentration of the latter may be, above a 
certain limit. 

According to the experiments of Euler and Johansson [1913] the 
hydrolytic activity of the hexosephosphatase is greatly diminished by 
the presence of toluene. 

Effect of Phosphate on the Total Fermentation Produced by 

Yeast-Juice. 

The addition of a phosphate to yeast-juice not only produces the 
effect already described, but also enables a given volume of yeast-juice 
to effect a larger total fermentation, even after allowance is made for 
the carbon dioxide equivalent to the quantity of phosphate added. 
The increase in the case of ordinary yeast-juice has been found to 
amount to from 10 to 150 per cent, of the original total fermentation 



THE FUNCTION OF PHOSPHATES 55 

produced by the juice in the absence of added phosphate. The numbers 
contained in columns I and 2 of the table on p. 56 illustrate this 
effect, the ratio of the total in the presence of phosphate to that 
obtained in its absence being given, as well as that of the total in pre- 
sence of phosphate less the equivalent of the phosphate added, to the 
original fermentation. The cause of this increase in the total ferment- 
ation is probably to be sought mainly in a protective action of the ex- 
cess of hexosephosphate on the various enzymes, for, as has been 
stated above, the rate of fermentation after the termination of the 
initial period, is practically the same as in the absence of added 
phosphate (see p. 43). 

Now it follows from equation (i) (p. 51) that in the total absence 
of phosphate no fermentation should occur, and the experimental 
realisation of this result would afford very strong evidence in favour 
of this interpretation of the phenomenon. 

Hitherto, however, it has not been found possible to free the 
materials employed completely from phosphorus compounds which 
yield phosphates by enzymic hydrolysis during the experiment, but it 
has been found that when the phosphate contents are reduced to as low 
a limit as possible, the amount of sugar fermented becomes correspond- 
ingly small, and, further, that in these circumstances the addition of a 
small amount of phosphate or hexosephosphate produces a relatively 
large increase in the fermenting power of the enzyme. 

When the total phosphorus present is thus largely reduced, the in- 
crease produced by the addition of a small amount of phosphate may 
amount to as much as eighty-eight times the original, in addition to the 
quantity equivalent to the phosphate, whilst the actual total evolved, 
including this equivalent, may be as much as twenty times the original 
fermentation. This result must be regarded as strong evidence in 
favour of the view that phosphates are indispensable for alcoholic fer- 
mentation. 

The results indicated above were experimentally obtained in three 
different ways and are exhibited in the following table. In the first 
place (cols. 3 and 4), advantage was taken of the fact that the residues 
obtained by filtering yeast-juice through a Martin gelatin filter (p. 59) 
are sometimes found to be almost free from mineral phosphates, whilst 
they still contain a small amount of co-enzyme. The experiment then 
consists in comparing the fermentation produced by such a residue poor 
in phosphate with that observed when a small amount of phosphate is 
added. The second method (col. 5) consisted in carrying out two 
parallel fermentations by means of a residue rendered inactive by fil- 



ALCOHOLIC FERMENTATION 



tration and a solution of co-enzyme free from phosphate and hexose- 
phosphate (p. 67) [Harden and Young, 1910, 2]. 

The third method (col. 6) consisted in washing zymin with water, 
to remove soluble phosphates, and then adding to it a solution of co- 
enzyme containing only a small amount of phosphate, and ascertaining 
the effect of the addition of a small known amount of hexosephosphate 
upon the fermentation produced by this mixture [Harden and Young, 
1911, 1} 





i 


2 


3 


4 


5 


6 


Gas evolved in absence of added phosphate . 
,, presence ,, ,, 


c.c. 

369 
629 


C.C. 

22O 
629 


C.C. 

i'4 
25-8 


C.C. 
I'2 

26-8 


C.C. 

20-3 

92-3 


C.C. 

i'5 
1327 


Increase due to phosphate .... 
Carbonic acid equivalent to phosphate . 


260 
63 


4 OQ 
6l 


24-4 
16-9 


25-6 
16-8 


72-0 
j.6-8 


131-2 


Increase after initial period .... 


197 


348 


7'5 


8-8 


55'2 





Ratio of totals 


I'7 


2'Q 


lS'4. 


21'3 


4'C 


88 


,, increase after initial period to 
original fermentation .... 


0'5 


r6 


5'3 


7*3 


2'7 





Production of a Fermentable Sugar from Hexosephosphate by 
the Action of an Enzyme Contained in Yeast- Juice. 

The sugar which, according to equation (2) accompanies the phos- 
phate formed by the enzymic hydrolysis of hexosephosphate is under or- 
dinary circumstances fermented by the alcoholic enzyme of the juice 
and thus escapes detection. 

When, however, a solution of a hexosephosphate is exposed to the 
action of either yeast-juice or zymin, entirely or partially freed from co- 
enzyme, this sugar, being no longer fermented, accumulates and can 
be examined. It has thus been found [Harden and Young, 1910, 2] 
that a sugar is in fact produced in this way which can be fermented by 
living yeast and exhibits the reactions of fructose, although the presence 
of other hexoses is not excluded. The products of the enzymic hydro- 
lysis of the hexosephosphates therefore appear to be the same as, or 
similar to, those formed by the action of acids [Young, 1909]. 

A further consequence of these facts is that a hexosephosphate will 
yield carbon dioxide and alcohol when it is added to yeast-juice or 
zymin, and this has also been found to be the case [Harden and Young, 
1910, 2; Iwanoff, 1909, i]. 



THE FUNCTION OF PHOSPHATES 57 

Mechanism of the Formation of Hexosediphosphoric Acid. 

On this subject little is yet known, but a number of extremely 
interesting results, the interpretation of which is still doubtful, have 
been obtained by Euler and his colleagues. Euler has obtained a 
yeast [Yeast H of the St. Erik's brewery in Stockholm] which differs 
from Munich yeast in several respects. A maceration extract prepared 
from the yeast dried at 40 in a vacuum produces no effect on a glucose 
solution containing phosphate. If, however, the glucose solution be 
previously partially fermented with living yeast and then boiled and 
filtered, the addition of the extract prepared from Yeast H brings 
about the esterification of phosphoric acid without any accompanying 
evolution of carbon dioxide [Euler and Ohlsn, 1911, 1912]. 

Euler interprets this as follows : (a) Glucose itself is not directly 
esterified, but must first undergo some preliminary change, which is 
brought about by the action of living yeast. No proof of the existence 
of a new modification of glucose in this solution has however been ad- 
vanced, other than its behaviour to extract of Yeast H, so that Euler's 
conclusion cannot be unreservedly accepted. It is moreover possible 
and even more probable that some thermostable catalytic substance 
(perhaps a co-enzyme) passes from the yeast into the glucose solution 
and enables the yeast extract to attack the glucose and phosphoric 
acid. A very small degree of esterification was also produced when 
an extract having no action on glucose and phosphate was added to 
glucose which had been treated with 2 per cent caustic soda for forty 
hours, but the nature of the compound formed was not ascertained 
[Euler and Johansson, 1912, 4]. (&) The esterification of phosphoric 
acid without the evolution of carbon dioxide implies that the enzyme 
by which this process is effected is distinct from that which causes the 
actual decomposition of the sugar. Euler goes further than this and 
regards the enzyme as a purely synthetic one, giving it the name of 
hexosephosphatese to distinguish it from the hexosephosphatase which 
hydro lyses the hexosephosphate. 

The evidence on which this conclusion is based cannot be regarded 
as satisfactory, inasmuch as it consists in the observation that in presence 
of sugar yeast extract does not hydrolyse the phosphoric ester. This, 
however, could not be expected since hydrolysis and synthesis under 
these conditions would ultimately proceed at equal rates. 

In any case the adoption of this nomenclature is inconsistent with 
the conception of an enzyme as a catalyst and is therefore inadvisable 
untiKthe reaction has been much more thoroughly studied. 



58 ALCOHOLIC FERMENTATION 

It may further be pointed out that no proof has yet been advanced 
that the phosphoric ester produced without evolution of carbon 
dioxide is identical with hexosediphosphoric acid produced with 
evolution of carbon dioxide. It is by no means improbable that it 
represents some intermediate stage in the production of the latter 
(see p. 117). 

Euler's other results on this subject may be briefly summarised as 
follows : 

(1) In presence of excess of sugar the esterification of the phos- 
phoric acid proceeds by a monomolecular reaction and is most rapid 
in faintly alkaline reaction [Euler and Kullberg, 1911, 3]. 

(2) When yeast extract has been heated for 30 minutes to 40 it 
effects the esterification of phosphoric acid at a much greater rate than 
the unheated extract (2-10 times). Heating at 50 for 30 minutes 
however completely inactivates the extract. The cause of the activa- 
tion is as yet unknown. The temperature coefficient for the unheated 
extract (i7'5-3O) is I '4-1*5 for 10 rise of temperature [Euler and 
Ohlsen, 1911]. 

(3) Yeasts which in the dried state all produce rapid esterification 
of phosphoric acid, yield extracts of very unequal powers in this 
respect [Euler, 1912, i]. 



CHAPTER IV. 

THE CO-ENZYME OF YEAST-JUICE. 

IN the previous chapter reference was made to the fact that the ad- 
dition of boiled yeast-juice greatly increases the amounts of carbon 
dioxide and alcohol formed from sugar by the action of a given volume 
of yeast-juice. 

When the boiled juice is dialysed the substance : or substances to 
which this effect is due pass into the dialysate, the residue being quite 
inactive. In order to ascertain the effect on the process of alcoholic 
fermentation of the complete removal of these unknown substances 
from yeast-juice itself, dialysis experiments were instituted with fresh 
yeast-juice, capable of bringing about an active production of carbon 
dioxide and alcohol from sugar. It was already known from the ex- 
periments of Buchner and Rapp [1898, i] that dialysis in parchment 
paper for seventeen hours at o against water or physiological salt 
solution only produced a diminution of about 20 per cent, in the 
total amount of fermentation obtainable, and in view of the less per- 
manent character of the juice from top yeasts a more rapid, method 
of dialysis was sought. This was found in the process of nitration 
under pressure through a film of gelatin, supported in the pores of a 
Chamberland filter candle, which had been introduced by Martin 

[1896]. 

In this way it was found possible to divide the juice into a residue 
and a filtrate, each of which was itself incapable of setting up the 
alcoholic fermentation of glucose, whereas, when they were reunited, 
the mixture produced almost as active a fermentation as the original 
juice [Harden and Young, 1905, I ; 1906, 2]. 

The apparatus employed for this purpose consists of a brass tube 
provided with a flange in which the gelatinised candle is held by a 
compressed india-rubber ring, and is shown in section in Fig. 5. Two 
such apparatus are used, each capable of holding about 70 c.c. of the 
liquid to be filtered. The tubes, after being filled with the yeast-juice, 
are connected by means of a screw joint with a cylinder of compressed 
air and the filtration carried out under a pressure of 50 atmospheres, 

59 



6o 



ALCOHOLIC FERMENTATION 




the arrangement employed being shown in Fig. 6. In the earlier ex- 
periments 25 to 50 c.c. of yeast-juice were placed in 
1 1 each tube and the filtration continued until no more 

ISl H 

liquid passed through. The residue was then washed 
several times in situ by adding water and forcing it 
through the candle. The time occupied in this process 
varied from six to twelve hours with different prepara- 
tions of yeast-juice. The candle was then removed 
from the brass casing and the thick, brown-coloured 
residue scraped off, dissolved in water, and at once 
examined. It was subsequently found to be possible 
to dry this residue in vacuo over sulphuric acid with- 
out seriously altering the fermenting power, and this 
led to a slight modification of the method, which is 
now conducted as follows. Two quantities of 50 c.c. 
each of yeast-juice are filtered, without washing, and 
the residues spread on watch-glasses and dried in 
vacuo. Two fresh quantities of 50 c.c. are then 
filtered through the same candles and the residues 
also dried. The 200 c.c. of juice treated in this way 
give a dry residue of 17 to 24 grams. The residue 
is then dissolved in 100 c.c. of water and filtered in 
quantities of 50 c.c. through two fresh gelatinised 
candles and the residue again dried. A considerable diminution in 
weight occurs, partly owing to incomplete removal from the candle 
and brass casing, and the final residue only amounts to about 9 to 1 2 
grams. Occasionally it is necessary to repeat the processes of dis- 
solving in water, filtering, and drying, but a considerable loss both of 
material and fermenting power attends each such operation. 

The sticky residue dries up very rapidly in vacuo to a brittle, scaly 
mass, which is converted by grinding into a light yellow powder. 

The filtrate was invariably found to be quite devoid of fermenting 
power, none of the enzyme passing through the gelatin. 

Properties of the Filtered and Washed Residue. The residue prepared 
as described above consists mainly of the protein, glycogen, and dex- 
trins of the yeast-juice, and is almost free from mineral phosphates, but 
contains a certain amount of combined phosphorus. It also contains 
the enzymes of the juice, including the proteoclastic enzyme, and the 
hexosephosphatase (p. 54). Its solution in water is usually quite 
inactive to glucose or fructose, but in some cases produces a small 
and evanescent fermentation. When the original filtrate or a corre- 



FIG. 5. 



THE CO-ENZYME OF YEAST-JUICE 



61 



spending quantity of the filtrate from boiled fresh yeast-juice is added, 
the mixture ferments glucose or fructose quite readily. The following 




FIG. 6. 

table shows the quantitative relations observed, the sugar being in all 
cases present in excess : 



No. 


Material. 


Volume. 


Filtrate 
added. 


Boiled Juice 
added. 


Water 
added. 


CO 2 evolved. 


I 


Undried and unwashed 














residue . 


15 cc. 


C.C. 


O C.C. 


15 c.c. 


o gram. 






15 


15 


o 


o ,, 


o'035 n 


2 


> 


15 


o 


o 


15 ,, 


0-024 ,, 






15 


o ,, 


15 


o ,, 


0-282 


3 


Undried and washed 














residue 


25 


o ,, 


o ,, 


o ,, 


0*4 c.c. 






25 


o ,, 


25 


o ,, 


268 


4 


> 


20 


o 


o 


o ,, 


8'3 i, 






2O 


20 


o ,, 


o ,, 


90'3 ii 


5 


Washed and dried re- 














sidue 


I gram in 














15 c.c. . 


o 


it 


o ,, 


o ,, 






t) 


o 


12 ,, 


o 


108 


6 


> 


i gram in 














25 c.c. . 


o 


O ,, 


o 


o ,, 






n 


o 


25 I, 


o 


364 >, 



62 ALCOHOLIC FERMENTATION 

These experiments lead to the conclusion that the fermentation of 
glucose and fructose by yeast-juice is dependent upon the presence, not 
only of the enzyme, but also of another substance which is dialysable 
and thermostable. 

Precisely similar results were subsequently obtained by Buchner 
and Antoni [1905, 2] by the dialysis of yeast-juice. One hundred 
c.c. of juice were dialysed for twenty- four hours at o against 1300 c.c. 
of distilled water, and the dialysate was then evaporated at 40 to 50 
to 20 c.c. The fermenting power of 20 c.c. of the dialysed juice was 
then determined with the following additions : 

(1) 20 c.c. of dialysed juice + 10 c.c. of water gave 0-02 gram CO 2 . 

(2) + 10 evaporated dialysate gave 0-52 gram CO 2 . 

(3) + 10 boiled juice gave 0-89 gram CO 2 . 

It was shown in the previous chapter that phosphates are essential 
to fermentation, and hence it becomes necessary to inquire whether 
the effect of dialysis is simply to remove these. Experiment shows 
that this is not the case. Soluble phosphates do not confer the power 
of producing fermentation on the inactive residue obtained by filtra- 
tion. Moreover, when yeast-juice is digested for some time before 
being boiled, it is found, as will be subsequently described, that the 
boiled autolysed juice is quite incapable of setting up fermentation in 
the inactive residue, although free phosphates are abundantly present 
[Harden and Young, 1906, 2]. 

The filtration residue is never obtained quite free from combined 
phosphorus, but the production from this of the phosphate necessary 
for fermentation to proceed, may be so slow as to render the test for 
co-enzyme uncertain, owing to the absence of sufficient phosphate. 
When a filtration residue is being tested it is therefore necessary to 
secure the presence of sufficient phosphate to enable the characteristic 
reaction to proceed, and at the same time to avoid adding phosphate 
in too great concentration, as this may, in the presence of only small 
amounts of enzyme or co-enzyme, inhibit the fermentation (p. 71). 
The proof that a filtration residue or dialysed juice is quite free from 
co-enzyme is therefore a somewhat complicated matter, and not only 
involves the experimental demonstration that the material will not 
ferment sugar, but also that this power is not imparted to it by the 
addition of a small concentration of phosphate. As it has been 
found (p. 73) that the fermentation of fructose is less affected than 
that of glucose by the presence of excess of phosphate, the practical 
method of examining a filtration residue for co-enzyme is to test its 
action on a solution of fructose (i) alone and (2) in presence of a 
small concentration of phosphate. If the residue produces no action 



THE CO-ENZYME OF YEAST-JUICE 63 

in either case, but produces fermentation when a solution of co-enzyme 
is added in the presence of the same concentration of phosphate as 
was previously employed, it may be concluded that this sample was 
free from co-enzyme but contained enzyme ; such an experiment also 
affords a definite proof that the co-enzyme does not consist of phos- 
phate. 

This dialysable, thermostable substance, without which alcoholic 
fermentation cannot proceed, has been provisionally termed the co- 
ferment or co-enzyme of alcoholic fermentation. This expression was 
first introduced by Bertrand [1897], to denote substances of this kind, 
and he applied it in two instances to the calcium salt which he con- 
sidered was necessary for the action of pectase on pecten substances, 
and to the manganese which he supposed to be essential for the activ- 
ity of laccase. Without inquiring whether these substances are pre- 
cisely comparable in function with that contained in yeast-juice, the 
term may be very well applied to signify the substance of unknown 
constitution without the co-operation of which the thermolabile enzyme 
of yeast-juice is unable to set up the process of alcoholic fermentation. 
The active agent of yeast-juice consisting of both enzyme and co-enzyme 
may be conveniently spoken of as the fermenting complex, and this 
term will occasionally be employed in the sequel. 

The co-enzyme is present alike in the filtrates from fresh yeast- 
juice and from boiled yeast-juice, and is also contained in the liquids 
obtained by boiling yeast with water and by washing zymin or dried 
yeast with water. 

Practically the only chemical property of the co-enzyme, other than 
that of rendering possible the process of alcoholic fermentation, which 
has so far been observed, is that it is capable of being decomposed, 
probably by hydrolysis, by a variety of reagents, prominent among 
which is yeast-juice. This was observed by Harden and Young in 
the course of their attempts to prepare a completely inactive residue 
by filtration. In many cases a residue was obtained which still pos- 
sessed a very limited power of fermentation, only a small amount of 
carbon dioxide being formed and the action ceasing entirely after 
the expiration of a short period ; on the subsequent addition of boiled 
juice, however, a very considerable evolution of carbon dioxide was 
produced. This was interpreted to mean that the residue in question 
contained an ample supply of enzyme but only a small proportion of 
co-enzyme, and that the latter was rapidly destroyed, so that the fer- 
mentation soon ceased. The boiled juice then added provided a further 
proportion of co-enzyme by the aid of which the surplus enzyme was 



64 ALCOHOLIC FERMENTATION 

enabled to carry on the fermentation. This view was confirmed by 
adding to a solution of a completely inactive filtration residue and 
glucose successive small quantities of boiled juice and observing the 
volumes of carbon dioxide evolved after each such addition. Thus in 
one case successive additions of volumes of 3 c.c. of boiled juice pro- 
duced evolutions of 8*2, 6, and 6 c.c. of carbon dioxide. In another 
case two successive additions of 1 5 c.c. of boiled juice produced evolu- 
tions of 54 and 41*2 c.c. On the other hand, the enzyme itself also 
gradually disappears from yeast-juice when the latter is incubated 
either alone or with sugar (p. 20). 

The cessation of fermentation in any particular mixture of enzyme 
and co-enzyme may, therefore, be due to the disappearance of either of 
these factors from the liquid. If the amount of co-enzyme present be 
relatively small it is the first to disappear, and fermentation can then 
only be renewed by the addition of a further quantity, whilst the 
addition of more enzyme produces no effect. If, on the other hand, 
the amount of co-enzyme be relatively large, the inverse is true ; the 
enzyme is the first to disappear, and fermentation can only be renewed 
by the addition of more enzyme, a further quantity of co-enzyme pro- 
ducing no effect. It has, moreover, been found that the co-enzyme, 
like the enzyme, disappears more rapidly in the absence of glucose 
than in its presence, incubation at 25 for two days being as a rule 
sufficient to remove all the co-enzyme from yeast-juice from top yeasts 
in the absence of sugar, whilst in the presence of fermentable sugar 
co-enzyme may still be detected at the end of four days. 

In all the experiments carried out by Harden and Young with 
juice from English top yeast it was found that when a mixture of the 
juice with glucose was incubated until fermentation had ceased, the 
further addition of co-enzyme in the form of boiled juice did not 
cause any renewal of the action ; in other words, the whole of the 
enzyme had disappeared. 

On the other hand, Buchner and Klatte [1908], working with juice 
and zymin prepared from bottom yeast, observed the extremely inter- 
esting fact that after the cessation of fermentation the addition of an 
equal volume of boiled juice caused a renewed decomposition of sugar, 
and that the processes of incubation until no further evolution of gas 
occurred and re-excitation of fermentation by the boiled juice could be 
repeated as many as six times. Thus in one experiment the duration 
of the fermentation was extended from three to a total of twenty-four 
days, and the total gas evolved from 073 gram to 2-19 grams. The 
phenomenon has been found to be common to yeast from Munich and 



THE CO-ENZYME OF YEAST-JUICE 65 

from Berlin as well as to zymin and maceration extract, and it was 
further observed that the boiled juice from one yeast could regenerate 
the juice from another, although the quantitative relations were 
different. 

In these samples of yeast-juice, therefore, there is present a natural 
condition of affairs precisely similar to that obtaining in the artificial 
mixtures of inactive filtration residue and co-enzyme solution made by 
Harden and Young. The balance of quantities is such that the co- 
enzyme disappears before the enzyme, leaving a certain amount of 
enzyme capable of exercising its usual function as soon as sufficient 
co-enzyme is added. This establishes an interesting point of contrast 
with the juice prepared from top yeast in England, in which the 
enzyme does not outlast the co-enzyme [Harden and Young, 1907]. 
The difference may be due to some variation in the relative propor- 
tions of enzyme and co-enzyme or of the enzymes to which the dis- 
appearance of each of these is presumptively due, or to a combination 
of these two causes. It was, however, found, even in the juice from 
bottom yeast, that incubation for three days at 22 without the addi- 
tion of sugar caused the disappearance of the enzyme as well as of the 
co-enzyme, and left a residue alike incapable of being regenerated by 
the addition of co-enzyme or of restoring the power of producing fer- 
mentation to an inactive mixture containing enzyme and sugar. 

If the fermenting power of the juice is to be preserved by repeated 
regeneration for a long period, it is absolutely necessary to add the 
co-enzyme solution each time as soon as fermentation has ceased, since 
the enzyme in the absence of this addition rapidly disappears, even 
in the presence of sugar. 

This result is probably to be explained, at all events in the main, by 
the presence in the co-enzyme solution of the antiprotease to which re- 
ference has already been made [Buchner and Haehn, 1910, 2]. This 
agent, the constitution of which is still unknown, protects proteins in 
general from the action of digestive enzymes, and on the assumption 
that the alcoholic enzyme of yeast-juice belongs to the class of proteins, 
may be supposed to lessen the rate at which this enzyme is destroyed 
by the endotryptase of the juice. This antiprotease is, like the co- 
enzyme (p. 68), destroyed by lipase but is more stable than the co- 
enzyme towards hydrolytic agents, and can be obtained free from 
co-enzyme by boiling the solution for some hours alone or by heating 
with dilute sulphuric acid. Such a solution possesses no regenerative 
power, but still retains its power of protecting proteins against digestion 
and of preserving the fermenting power of yeast-juice, 

5 



66 ALCOHOLIC FERMENTATION 

It must, however, be remembered that the addition of a phosphate 
alone may greatly prolong the period of fermentation of yeast-juice 
(p. 55), and sugar is well known to exert a similar action. It appears, 
therefore, that the existence of the enzyme is prolonged not only by 
the presence of the antiprotease but also by that of sugar and hexose- 
phosphate, into which phosphate passes in presence of sugar. Similar 
effects are exerted on the co-enzyme by sugar and probably also by 
hexosephosphate. 

The fermenting complex, therefore, in the presence of these sub- 
stances, either separately or together, falls off more slowly in activity 
and is present for a longer time, and for both of these reasons produces 
an increased amount of fermentation. It seems probable also that the 
hexosephosphatase is similarly affected, so that the supply of free 
phosphate is at the same time better maintained, and the rate of fer- 
mentation for this reason decreases more slowly than would otherwise 
be the case. 

It is in this way that an explanation may be found of the remark- 
able increase in total fermentation, which is produced by the addition 
to yeast-juice and sugar of boiled yeast-juice, containing free phos- 
phate (which passes into hexosephosphate) as well as co-enzyme, of 
boiled autolysed yeast-juice, containing free phosphate but no co- 
enzyme, or of phosphate solution alone. 

In no case is the original rate of fermentation greatly increased 
after the initial acceleration has disappeared, but in every case the 
total fermentation is considerably augmented, and this is no doubt 
mainly to be attributed, as just explained, to the diminished rate of 
decomposition of the fermenting complex and probably of the hexose- 
phosphatase. 

Although both enzyme and co-enzyme are completely precipitated 
from yeast-juice, as already described (p. 38), by 10 volumes of acetone, 
the co-enzyme is less easily precipitated than the enzyme, and a certain 
degree of separation can therefore be attained by fractional precipit- 
ation [Buchner and Duchac'ek, 1909]. The enzyme cannot, how- 
ever, be completely freed from co-enzyme in this manner, and the process 
is attended by a very considerable loss of enzyme. This is probably 
due to the fact that only small quantities of acetone can be added (i '5 
to 3 volumes), in order to avoid precipitation of co-enzyme, and that 
the precipitates thus formed contain a large proportion of water, a con- 
dition which appears to be fatal to the preservation of the enzyme. 

It is, however, not quite certain whether it is the zymase or the 
hexosephosphatase which is destroyed in these cases, as no attempt 



THE CO-ENZYME OF YEAST-JUICE 67 

was made to distinguish between them. In any case the precipitates 
obtained by fractional treatment with acetone, even when reunited, 
produce a much smaller fermentation than the original juice or the 
powder prepared by bringing it into 10 volumes of acetone. 

Attempts to isolate the co-enzyme from boiled yeast-juice have also 
been hitherto unsuccessful. It has, however, been found possible to 
remove a considerable amount of material from the solution without 
affecting the co-enzyme. When I volume of alcohol is added to 
boiled yeast-juice, a bulky precipitate, consisting largely of carbo- 
hydrates, is produced, and the filtrate from this is found to contain the 
co-enzyme and can be freed from alcohol by evaporation. Further 
precipitation with alcohol has not led to useful results. 

When a solution which has been treated in this way is precipitated 
with lead acetate and kept neutral to litmus, the free phosphate and 
hexosephosphate are thrown down and the co-enzyme remains in solu- 
tion. The filtrate can be freed from lead by means of sulphuretted 
hydrogen and neutralised, and then forms a solution of co-enzyme free 
from phosphate and hexosephosphate but still containing combined 
phosphorus. More complete purification than this has not yet been 
accomplished. Occasionally the precipitate of lead salts retains some 
of the co-enzyme, apparently by adsorption, but usually the greater 
part remains in the solution (Harden and Young). 

The co-enzyme is partially removed from yeast-juice by means of 
a colloidal solution of ferric hydroxide (Resenscheck). A precipitate 
is thus obtained which contains phosphorus and resembles boiled yeast- 
juice in its regenerative action on yeast-juice rendered inactive by fer- 
mentation. It has not, however, so far been found possible to isolate 
any definite compound from this precipitate. There are also indications 
that when yeast-juice, either fresh or boiled, is electrolysed, the co- 
enzyme tends to accumulate at the cathode [Resenscheck, 1908, I, 2]. 

Buchner and Klatte [1908] made use of yeast-juice rendered free 
from co-enzyme by incubation with sugar solution to examine the 
nature of the agent by which the co-enzyme is destroyed. This agent 
is certainly an enzyme, since boiled yeast-juice can be preserved with 
unimpaired powers for a considerable length of time, and suspicion fell 
naturally, in the first instance, on the endotryptase of the yeast cell. 
Direct experiment showed, however, that yeast-juice, which, when fresh, 
rapidly destroyed the co-enzyme of boiled juice, lost this power on 
preservation, but retained its proteoclastic properties without diminu- 
tion, so that the tryptic enzyme could not be the one concerned. The 
direct action of commercial trypsin on boiled yeast-juice also yielded 

5* 



68 ALCOHOLIC FERMENTATION 

a negative result, although this cannot strictly be regarded as an indic- 
ation of the effect of the specific proteoclastic enzymes of yeast-juice. 
On the other hand, it was found that when boiled juice was treated 
for some time with an emulsion containing the lipase of castor oil seeds, 
the co-enzyme was completely destroyed. This is a result of great im- 
portance, inasmuch as it probably indicates that the co-enzyme is 
chemically allied to the class of substances hydrolysable by lipase, i.e. 
to the fats and other esters. 

Further, observations by Buchner and Haehn [1909] have shown 
that digestion with potassium carbonate solution containing 2*5 grams 
per 100 c.c. also brings about the destruction of the co-enzyme, and that 
this is also slowly accomplished by the repeated boiling of the juice. The 
co-enzyme is also destroyed both by acid and alkaline hydrolysis, and 
when the solution is evaporated to dryness and the residue ignited. 

Beyond this general indication nothing is known of the chemical 
nature of the co-enzyme. The intimate relation of phosphoric acid to 
the process of fermentation renders it not impossible that the co- 
enzyme may contain this group, but there is no definite evidence for 
such a belief. Purely negative results have been obtained with all the 
substances of known composition which have yet been tested, among 
these being soluble phosphates, hexosephosphates and a number of 
oxidisable and reducible substances, such as quinol, p-phenylene- 
diamine, methylene blue, peptone beef broth, etc. (Harden and Young ; 
Harden and Norris [1914]; see also Euler and Backstrom [1912]), 
glycero-phosphates (Buchner and Klatte). 

The precise function of the co-enzyme is even more obscure than 
its chemical nature. The system of reacting substances consisting of 
fermentable material, enzyme and co-enzyme, bears, however, an obvious 
superficial resemblance to many of the systems required for the accom- 
plishment of chemical changes in the animal or vegetable organism. 
Such a triad of substances is, for example, requisite for the process by 
which the red blood corpuscles of an animal are broken up by the 
serum of a different animal into the blood of which the red corpuscles 
of the first animal have been injected. This effect is only produced 
when two substances are present, the amboceptor or immune body and 
the complement. The analogy may be carried to a further stage since 
the amboceptor is, like the co-enzyme, more thermostable than the 
complement, which therefore corresponds with the enzyme. Immune 
serum can, in fact, be freed from complement by being heated at 57-60 
for half an hour, whereas the amboceptor is unaffected by this treat- 
ment. On the other hand, the complement and amboceptor do not 



THE i CO-ENZYME OF YEAST-JUICE 69 

appear to act like enzymes but rather like ordinary chemical 
reagents, remaining in combination even after the blood corpuscle has 
been broken up, whereas the enzyme and co-enzyme of yeast-juice 
are again liberated when the reaction between sugar and phosphate 
has been completed. 



CHAPTER V. 

ACTION OF SOME INHIBITING AND ACCELERATING AGENTS ON THE 
ENZYMES OF YEAST-JUICE. 

ONE of the most interesting and at the same time most difficult 
problems concerning enzyme action in general is the nature of the 
inhibiting or accelerating effect produced by many substances upon 
the rate or total result of the chemical process set up in presence of the 
enzyme. Inhibition, it is usually supposed, involves either the de- 
composition of the enzyme, in which case it is irreversible, its removal 
from the sphere of action by some change in its mode of solution, or 
the formation of an inactive or less active compound between the 
enzyme and the inhibiting agent. This compound it may sometimes 
be possible to decompose, with the result that the activity of the enzyme 
is restored. A striking example of this, to which allusion has already 
been made, is the effect of hydrocyanic acid on alcoholic fermentation 

(P- 37). 

Acceleration of enzyme action can in some cases be ascribed to the 
fact that the accelerating substance possesses an assignable chemical 
function in the reaction, so that an increase in the concentration of this 
substance causes an increase in the rate of the reaction. As we have 
seen in Chapter III, this is the explanation of the accelerating effect 
of phosphates on fermentation by yeast-juice. In many other cases, 
however, no such chemical function can be traced, as, for example, in 
the effect of neutral salts on the hydrolytic action of invertase, or the 
effect of the addition of the co-enzyme to zymase, and it is necessary to 
fall back on some assumption, such as that the accelerating agent acts 
by increasing the effective concentration of the enzyme or by com- 
bining either with the enzyme or the substrate, forming a compound 
which undergoes the reaction more readily. 

The interest in the following examples of inhibition and acceleration 
of fermentation by yeast-juice lies not only in their relation to these 
general problems but also, and perhaps chiefly, in their bearing on the 
specific problem of the nature and mode of action of the various agents 
concerned in the production of alcohol and carbon dioxide from sugar 
in the yeast-cell. 

70 



ACTION OF INHIBITING AND ACCELERATING AGENTS 71 

I. Influence of Concentration of Phosphate on the Course of 

Fermentation. 

Prominent among these instances of inhibition and acceleration are 
the phenomena attendant on the addition of excess of phosphate to 
yeast-juice. 

When a phosphate is added to a fermenting mixture of a sugar 
and yeast-juice, the effect varies with the concentration of the phos- 
phate and the sugar and with the particular specimen of yeast-juice 
employed. With low concentrations of phosphate in presence of excess 
of glucose the acceleration produced is so transient that no accurate 
measurements of rate can be made. As soon as the amount of phos- 
phate added is sufficiently large, it is found that the rate of evolution 
of carbon dioxide very rapidly increases from five to ten times, and 
then quickly falls approximately to its original value. 

As the concentration of phosphate is still further increased, it is 
first observed that the maximum velocity, which is still attained almost 
immediately after the addition of the phosphate, is maintained for a 
certain period before the fall commences, and then, as the increase in 
concentration of phosphate proceeds, that the maximum is only 
gradually attained after the addition, the period required for this in- 
creasing with the concentration of the phosphate. Moreover, with 
still higher concentrations, the maximum rate attained is less than that 
reached with lower concentrations, and further, the rate falls off more 
slowly. The concentration of phosphate which produces the highest 
rate, which may be termed the optimum concentration, varies very 
considerably with different specimens of yeast-juice [Harden and 
Young, 1908, i]. 

All these points are illustrated by the accompanying curves (Fig. 7) 
which show the rate of evolution per five minutes plotted against the 
time for four solutions in which the initial concentrations of phosphate 
were (A) 0-033, (B) 0-067, (C)o'i, and (D) 0-133 molar, the volumes of 
0-3 molar phosphate being 5, 10, I 5, and 20 c.c. in each case added to 
25 c.c. of yeast-juice, and made up to 45 c.c., each solution containing 
4 '5 grams of glucose. The time of addition is taken as zero, the rate 
before addition being constant, as shown in the curves. 

It will be observed that 5 and 10 c.c. (A and B) give the same 
maximum, whilst 15 c.c. (C) produce a much lower maximum, and 20 
c.c. (D) a still lower one, the rate at which the velocity diminishes 
after the attainment of the maximum being correspondingly slow in 
these last two cases. By calculating the amount of phosphate which 
has disappeared as such from the amount of carbon dioxide evolved, 



Jl ALCOHOLIC FERMENTATION 

it is found that the maximum does not occur at the same concentration 
of free phosphate in each case. 

These results suggest that the phosphate is capable of forming 
two or more different unstable associations with the fermenting com- 
plex. One of these, formed with low concentrations of the phosphate, 
has the composition most favourable for the decomposition of sugar, 
whilst the others, formed with higher concentrations of phosphate, 
contain more of the latter, probably associated in such a way with the 
fermenting complex as to render the latter partially or wholly incapable 
of effecting the decomposition of the sugar molecule. As the fermenta- 




o 5 ID 15 20 25 50 
Time in minutes 



tion proceeds slowly in the presence of excess of phosphate, the concen- 
tration of the latter is reduced by conversion into hexosephosphate, 
and a re-distribution of phosphate occurs, resulting in the gradual 
change of the less active into the more active association of phosphate 
with fermenting complex, and a consequent rise in the rate of fermen- 
tation. 

In those cases in which the maximum rate corresponding to the 
optimum concentration of phosphate is never attained, some secondary 
cause may be supposed to intervene, such as a permanent change in 
a portion of the fermenting complex, accumulation of the products of 
the reaction, etc. 

It is also possible as suggested by Buchner for the analogous case 
of arsenite (p. 78) that the addition of increasing amounts of phos- 
phate causes a progressive but reversible change in the mode of dis- 



ACTION OF INHIBITING AND ACCELERATING AGENTS 73 



persion of the colloidal enzyme, and that this has the secondary effect 
of altering the rate of fermentation. No decisive evidence is as yet 
available upon the subject. 

The results obtained by Euler and Johansson [1913] to which 
reference has already been made indicate that in presence of a 
moderate excess of phosphate esterification is more rapid than pro- 
duction of carbon dioxide. No explanation of this phenomenon has 
yet been given, but it might obviously be due either to the production 
of some phosphorus compound which subsequently takes part in the 
production both of hexosediphosphate and of carbon dioxide, or, less 
probably, to the entire independence of the two changes esterification 
of phosphate and production of carbon dioxide which might then be 
differently affected by the presence of excess of phosphate and there- 
fore take place at different rates. 

II. Reaction of Fructose with Phosphates in Presence of 

Yeast- Juice. 

Although, as has been pointed out (p. 42), glucose, mannose, and 
fructose all react with phosphate in a similar manner in presence of 
yeast-juice, there are nevertheless certain quantitative differences be- 
tween the behaviour of glucose and mannose on the one hand, and 
fructose on the other, which appear to be of considerable importance. 
Fructose differs from the other two fermentable hexoses in two par- 
ticulars : (i) the optimum concentration of phosphate is much 
greater; (2) the maximum rate of fermentation attainable is much 
higher [Harden and Young, 1908, 2 ; 1909]. 

These points are clearly illustrated by the following results, which 
all refer to 10 c.c. of yeast-juice, and show that the optimum concen- 
tration of phosphate for the fermentation of fructose is from 1*5 to 
10 times that of glucose, and that the maximum rate of fermentation 
for fructose in presence of phosphate is 2 to 6 times that of glucose. 



Sugar, in 
Grams. 


Total 
Volume. 


Optimum Volume of 
0'6 Molar Phosphate in c.c. 


Maximum Rate in Cubic 
Centimetres of COg per 
Five Minutes. 






Glucose. 


Fructose. 


Glucose. 


Fructose. 


2 


35 


2 


5 


7'5 


32'2 


4 


50 


' I 


10 


5*4 


28-4 


1-6 


23 


2 


5 


8 


17 


i 


25 


175 


5 


5'2 


25-9 


2 


25 


5 


7*5 


16-2 


31-2 


2 


20 


2 


3'5 


7'9 


22'6 


2 


22'5 


075 


2 


3'4 


22'2 



74 ALCOHOLIC FERMENTATION 

It is interesting to note that the two high rates, 32*2 and 31 -2 c.c. 
per five minutes, are equal to about half the rate obtainable with an 
amount of living yeast corresponding to 10 c.c. of yeast-juice, assum- 
ing that about 1 6 to 20 grams of yeast are required to yield this 
volume of juice, and that this amount of yeast would give about 56 
to 70 c.c. of carbon dioxide per five minutes at 25, which has been 
found experimentally to be about the rate obtainable with the top 
yeast employed for these experiments. 

III. Effect of the Addition of Fructose on the Fermentation of 
Glucose or Mannose in Presence of a Large Excess of 
Phosphate. 

When the maximum rate of fermentation of glucose or mannose 
by yeast-juice in presence of phosphate is greatly lowered by the 
addition of a large excess of phosphate, the addition of a relatively 
small amount of fructose (as little as 2-5 per cent, of the weight of the 
glucose) causes rapid fermentation to occur. This induced activity is 
not due solely to the selective fermentation of the added fructose, since 
the amount of gas evolved may be greatly in excess of that obtainable 
from the quantity added. 

Another way of expressing the same thing is to say that the 
optimum concentration of phosphate (p. 71) is greatly raised when 
2 -5 per cent, of fructose is added to glucose, and that consequently the 
rate of fermentation rises. The effect is extremely striking, since a 
mixture of glucose and yeast-juice fermenting in the presence of a 
large excess of phosphate at the rate of less than I c.c. of carbon 
dioxide in five minutes may be made to ferment at six to eight times 
this rate by the addition of only 0*05 gram of fructose (2-5 per cent, 
of the glucose present), and to continue until the total gas evolved is at 
least five to six times as great as that obtainable from the added fructose, 
the concentration of the phosphate being the whole time at such a 
height as would limit the fermentation of glucose alone to its original 
value. 

The effect is not produced when the concentration of the phosphate 
is so high that the rate of fermentation of fructose is itself greatly 
lowered. 

This remarkable inductive effect is specific to fructose and is not 
produced when glucose is added to mannose or fructose, or by mannose 
when added to glucose or fructose, under the proper conditions of 
concentration of phosphate in each case. 

This interesting property of fructose, taken in connection with the 



ACTION OF INHIBITING AND ACCELERATING AGENTS 75 

facts that this sugar in presence of phosphate is much more rapidly 
fermented than glucose or mannose, and that the optimum concentration 
of phosphate for fructose is much higher than for glucose or mannose, 
appears to indicate that fructose when added to yeast-juice does not 
merely act as a substance to be fermented, but in addition, bears some 
specific relation to the fermenting complex. 

All the phenomena observed are, indeed, consistent with the sup- 
position that fructose actually forms a permanent part of the fermenting 
complex, and that, when the concentration of this sugar in the yeast- 
juice is increased, a greater quantity of the complex is formed. As the 
result of this increase in the concentration of the active catalytic agent, 
the yeast-juice would be capable of bringing about the reaction with 
sugar in presence of phosphate at a higher rate, and at the same time 
the optimum concentration of phosphate would become greater, exactly 
as is observed. The question whether, as suggested above, fructose 
actually forms part of the fermenting complex, and the further questions, 
whether, if so, it is an essential constituent, or whether it can be replaced 
by glucose or mannose with formation of a less active complex, remain 
at present undecided, and cannot profitably be more fully discussed until 
further information is available. 

It must, moreover, be remembered that different samples of yeast- 
juice vary to a considerable extent in their relative behaviour to glucose 
and fructose, so that the phenomena under discussion may be expected 
to vary with the nature and past history of the yeast employed. 

IV. Effect of Arsenates on the Fermentation of Sugars by 
Yeast-Juice and Zymin. 

The close analogy which exists between the chemical functions of 
phosphorus and arsenic lends some interest to the examination of the 
action of sodium arsenate upon a mixture of yeast-juice and sugar, and 
experiments reveal the fact that arsenates produce a very considerable 
acceleration in the rate of fermentation of such a mixture [Harden and 
Young, 1906, 3; 1911, i]. The phenomena observed, however, differ 
markedly from those which accompany the action of phosphate. 

The acceleration produced is of the same order of magnitude as 
that obtained with phosphate, but it is maintained without alteration 
for a considerable period, so that there is no equivalence between the 
amount of arsenate added and the extra amount of fermentation effected. 
Further, no organic arsenic compound corresponding in composition 
with the hexosephosphates appears to be formed. 

Increase of concentration of arsenate produces a rapid inhibition of 



ALCOHOLIC FERMENTATION 



fermentation, probably due to some secondary effect on the fermenting 
complex, possibly to be interpreted as the formation of compounds in- 
capable of combining with sugar and hence unable to carry on the pro- 
cess of fermentation. An optimum concentration of arsenate therefore 
exists just as of phosphate, at which the maximum rate is observed, 
and this optimum concentration and the corresponding rate vary with 
different samples of juice and are less for glucose than for fructose. 
The rate of fermentation by zymin is relatively less increased than that 
by yeast-juice. 

Owing to the fact that the rate is permanently maintained the ad- 
dition of a suitable amount of arsenate increases the total fermentation 
produced to a much greater extent than phosphate. 

The nature of these effects may be gathered from the result of a 
few typical experiments. In one case the rate of fermentation of 
glucose by yeast-juice was raised by the presence of 0*03 molar arsenate 
from 2 to 23 c.c. per five minutes, and the total evolved in ninety- 
five minutes from 51 10459 c.c. The accelerating effect on 20 c.c. of 
juice, of as little as 0*005 c - c - f '3 molar arsenate, containing 0*11 
mgrm. of arsenic, can be distinctly observed, but the maximum effect 
is usually produced by about I to 3 c.c., the concentration being 
therefore 0*015 to 0*045 rnolar. Greater concentrations than this pro- 
duce a less degree of acceleration accompanied by a shorter duration 
of fermentation, as shown by the following numbers which refer to 20 
c.c. of yeast-juice in a total volume of 40 c.c. containing 10 per cent, 
of glucose : 



C.cs. of 
0-3 Molar 
Arsenate 
in 40 c.c. 


Molar 
Concentration of 
Arsenate. 


Maximum 
Rate of 
Fermentation. 


O 


o 


3'5 


0-005 


0*0000375 


6-3 


O'OI 


0-000075 


8 


0'02 


0*00015 


14-2 


0*04 


0-0003 


19-9 


0-1 


0-00075 


29-7 


O'2 


0-0015 


35 


o*5 


0-00375 


34*9 


I'O 


0-0075 


29*5 


2'O 


0*015 


23*2 


5*o 


0*0375 


14*5 


I0'0 


0-075 


87 


15-0 


0-1125 


5'3 


20 


0-15 


3*2 



The contrast between glucose and fructose in their relations to 



ACTION OF INHIBITING AND ACCELERATING AGENTS 77 



arsenate are well exhibited in the following table, in which the rates of 
fermentation produced by arsenate in presence of excess of glucose 
and fructose respectively are given : 





Rate. 








Glucose. 


Fructose. 


croo75 molar 


I2'I 


26-6 


0-0225 (opt. for glucose) 


I3'4 





0*0525 (opt. for fructose) 




45-8 


0-1125 


5'I 


39 



Here the optimum concentration for fructose is more than twice 
that for glucose, whilst the maximum rate of fermentation obtainable 
with fructose is between three and four times the maximum given by 
glucose. 

V. Effect of Arsenites on the Fermentation Produced by 

Yeast-Juice. 

Effects somewhat similar to those produced by arsenates were 
observed by Buchner [Buchner and Rapp, 1897 ; 1898, I, 2, 3 ; 1899, 2 ; 
Buchner, E. and H., and Hahn, 1903, pp. 184-205] when potassium 
arsenite was added to yeast-juice. This substance, the action of which 
on yeast had been adduced by Schwann as a proof of the vegetable nature 
of this organism, was employed by Buchner on account of its poisonous 
effect on vegetable cells as an antiseptic and as a means of testing for 
the protoplasmic nature of the agent present in yeast-juice. Its effect 
on the fermentation was, however, found to be irregular, and at the 
same time it did not act as an efficient antiseptic in the concentrations 
which could be employed. Even 2 per cent, of arsenious oxide, added as 
the potassium salt, had in many cases a decided effect in diminishing the 
total fermentation obtained with cane sugar, and this effect increased 
with the concentration. A number of irregularities were also observed 
which cannot here be discussed It was further found that in some 
cases 2 per cent, of arsenious oxide inhibited the fermentation of 
glucose but not of saccharose, or of a mixture of glucose and fructose, 
whilst its effect on fructose alone was of an intermediate character. 

The important observation was also made by Buchner that the 
addition of a suitable quantity of arsenite as a rule caused a greatly 
increased fermentation during the first sixteen hours even in experiments 
in which the total fermentation was diminished. By examining the 
effect of arsenite on fermentation in a similar manner to that of arsenate, 
Harden and Young [1911, i] have found that a close analogy exists 



78 ALCOHOLIC FERMENTATION 

between the effects and modes of action of these substances, but that 
arsenite produces a much smaller acceleration than arsenate. An 
optimum concentration of arsenite exists, just as in the case of arsenate, 
which produces a maximum rate of fermentation. Further increase in 
concentration leads to inhibition, and in no case is there any indication 
of the production of an exactly equivalent amount of fermentation as 
in the case of phosphate. In various experiments with dialysed, 
evaporated, and diluted yeast-juice in which 2 per cent, of arsenious 
oxide was found by Buchner to inhibit fermentation, it is probable 
that, owing to the small amount of fermenting complex left, this 
amount of arsenious oxide was considerably in excess of the optimum 
concentration, although Buchner ascribes the effect to the removal of 
some of the protective colloids of the juice, owing to the prolonged 
treatment to which it had been subjected. 

The extent of the action of arsenite appears from the following re- 
sults. In one case a rate of I 7 c.c. was increased to 7 c.c. by cro6 molar 
arsenite. In another experiment it was found that the optimum con- 
centration was 0*04 molar arsenite, the addition of which increased the 
rate three-fold. As in the case of arsenate the optimum concentration 
and the corresponding maximum rate of fermentation are considerably 
greater for fructose than for glucose. The relative rates produced by the 
addition of equivalent amounts of arsenate and arsenite (i c.c. of O'3 
molar solution in each case to 20 c.c. of yeast-juice) were 27-5 and 3-1, 
the original rate of the juice being I 7. In general the optimum con- 
centration of arsenite is considerably greater than that of arsenate. 

The inhibiting effects of higher concentrations of arsenite and 
arsenate also present close analogies, but this most interesting aspect 
of the question has not yet been sufficiently examined to repay detailed 
discussion. Buchner [Buchner, E. and H. , and Hahn, 1 903, pp. 1 99-205 ] 
has suggested that the inhibition is due primarily to some change in 
the colloidal condition of the enzyme and has shown that certain 
colloidal substances appear to protect it, as does also sugar. The 
possibility is also present that inactive combinations of some sort are 
formed between the fermenting complex and the inhibiting agent, in 
the manner suggested to account for the inhibiting effect of excess of 
phosphate (p. 72). It seems most probable that the effect is a com- 
plex one, in which many factors participate. 

Nature of the Acceleration Produced by Arsenate and Arsenite. 

In explanation of the remarkable accelerating action of arsenates 
and arsenites two obvious possibilities present themselves. In the 



ACTION OF INHIBITING AND ACCELERATING AGENTS 79 

first place the arsenic compound may actually replace phosphate in the 
reaction characteristic of alcoholic fermentation, the resulting arsenic 
analogue of the hexosephosphate being so unstable that it undergoes 
immediate hydrolysis, and is therefore only present in extremely small 
concentration at any period of the fermentation and cannot be isolated. 
In the second place it is possible that the arsenic compound may ac- 
celerate the action of the hexosephosphatase of the juice, and thus 
by increasing the rate of circulation of the phosphate produce the 
permanent rise of rate. With this effect may possibly be associated 
a direct acceleration of the action of the fermenting complex. 

The experimental decision between these alternative explanations 
is rendered possible by the use of a mixture of enzyme and co-enzyme 
free from phosphate and hexosephosphate. As has already been 
described (p. 55) a mixture of boiled yeast-juice, which has been 
treated with lead acetate, glucose or fructose, and washed zymin can 
be prepared which scarcely undergoes any fermentation unless phosphate 
be added. If now arsenates or arsenites can replace phosphate, they 
should be capable of setting up fermentation in such a mixture. 
Experiment shows that they do not possess this power. For ferment- 
ation to proceed phosphate must be present and it cannot be replaced 
either by arsenate or arsenite [Harden and Young, 1911, ij 

The effect of these salts on the action of the hexosephosphatase 
can also be ascertained by a modification of the foregoing experiment. 
If a hexosephosphate be made the sole source of phosphate in such a 
mixture as that described above, in which it must be remembered 
abundance of sugar is present, the rate at which fermentation can 
proceed will be controlled by the rate at which the hexosephosphate is 
decomposed with formation of phosphate. Experiment shows that in 
the presence of added arsenate or arsenite the rate of fermentation is 
largely increased, so that the effect of these salts must be to increase 
the rate of liberation of phosphate, or in other words, to accelerate the 
hydrolytic action of the hexosephosphatase. 

This conclusion is even more strikingly confirmed by a comparison 
of the direct action of yeast-juice on hexosephosphate in presence and 
in absence of arsenate, as measured by the actual production of free 
phosphate. In a particular experiment this gave rise to 0^0707 
gram of Mg 2 P 2 O 7 in the absence of arsenate and O'6i36 gram of 
Mg 2 P 2 O 7 in the presence of arsenate. 

The results obtained with arsenite are throughout very similar to 
those given by arsenate, but are not quite so striking. It may there- 
fore be affirmed with some confidence that the chief action of arsen- 



80 ALCOHOLIC FERMENTATION 

ates and arsenites in accelerating the rate of fermentation of sugars by 
yeast-juice or zymin, consists in an acceleration of the rate at which 
phosphate is produced from the hexosephosphate by the action of 
the hexosephosphatase. 

It has further been found that arsenates, and to a less degree arsenites, 
also produce an acceleration of the rate of autofermentation of yeast- 
juice and of the rate at which glycogen is fermented. This turns out to 
be due in all probability to an increase in the activity of the glycogenase 
by the action of which the sugar is supplied which is the direct subject of 
fermentation. Thus in one case an initial rate of fermentation of gly- 
cogen of I -9 c.c. per five minutes was increased by 0-05 molar arsenate 
to 9 '7 and the amount of carbon dioxide evolved in two hours from 
38 to 158 c.c. Even this enhanced production of glucose from glyco- 
gen, however, is not nearly sufficient for the complete utilisation of the 
phosphate also being liberated by the action on the hexosephosphatase, 
for the addition of an excess of sugar produces a much higher rate, 
in this case 36 c.c. per five minutes. The effect of arsenate on the rate 
of action of the glycogenase seems therefore to be much smaller than 
on that of the hexosephosphatase. 

No other substances have yet been found which share these inter- 
esting properties with arsenates and arsenites, and no advance has 
been made towards an understanding of the mechanism of the acceler- 
ating action of these salts on the specific enzymes which are affected 
by them. 



CHAPTER VI. 

CARBOXYLASE. 

AN observation of remarkable interest, which promises to throw 
light on several important features of the biochemistry of yeast, was 
made in 1911, and has since then formed the subject of detailed 
investigation by Neuberg and a number of co-workers. 

It was found that yeast had the power of rapidly decomposing 
a large number of hydroxy- and keto-acids [Neuberg and Hildesheimer, 
1911 ; Neuberg and Tir, 1911 ; see also Karczag, 1912, I, 2]. The 
most important among these are pyruvic acid, CH 3 *COCOOH, and a 
considerable number of other aliphatic a-keto-acids which are decom- 
posed with evolution of carbon dioxide and formation of the corres- 
ponding aldehyde : 

R-CO-COOH = R-CHO + C0 2 . 

The reaction is produced by all races of brewer's yeast which 
have been tried, as well as by active yeast preparations and extracts 
and by wine yeasts [Neuberg and Karczag, 1911,4; Neuberg and 
Kerb, 1912, 2], The phenomenon can readily be exhibited as a 
lecture experiment by shaking up 2 g. of pressed yeast with 12 c.c. of 
I per cent, pyruvic acid, placing the mixture in a Schrotter's ferment- 
ation tube, closing the open limb by means of a rubber stopper 
carrying a long glass tube and plunging the whole in water of 38-40. 
Comparison tubes of yeast and water and yeast and I per cent, 
glucose may be started at the same time, and it is then seen that glucose 
and pyruvic acid are fermented at approximately the same rate 
[Neuberg and Karczag, 1911, i]. If English top yeast be used it is 
well to take 0*5 per cent, pyruvic acid solution and to saturate the liquids 
with carbon dioxide before commencing the experiment. The pro- 
duction of acetaldehyde can be readily demonstrated by distilling the 
mixture at the close of fermentation and testing for the aldehyde 
either by Rimini's reaction (a blue coloration with diethylamine and 
sodium nitroprusside) or by means of p-nitrophenylhydrazine which 
precipitates the hydrazone, melting at 128-5 [Neuberg and Karczag, 
1911, 2, 3]. 

81 6 



82 ALCOHOLIC FERMENTATION 

As the result of quantitative experiments it has been shown that 
80 per cent, of the theoretical amount of acetaldehyde can be recovered. 
The salts of the acids are also attacked, the carbonate of the metal, 
which may be strongly alkaline, being formed. Thus taking the case 
of pyruvic acid, the salts are decomposed according to the following 
equation : 



Under these conditions a considerable portion of the aldehyde 
undergoes condensation to aldol [Neuberg, 1912] : 

2 CH 3 CHO = CH 3 . CH(OH) CH 2 CHO. 

This change appears to be due entirely to the alkali and not to 
an enzyme since the aldol obtained yields inactive /8-hydroxybutyric 
acid on oxidation [Neuberg and Karczag, 1911, 3; Neuberg, 1912]. 
The various preparations derived from yeast which are capable of 
producing alcoholic fermentation also effect the decomposition of 
pyruvic acid in the same manner as living yeast. They are, however, 
more sensitive to the acidity of the pyruvic acid, and it is therefore 
advisable to employ a salt of the acid in presence of excess of a weak 
acid, such as boric or arsenious acid, which decomposes the carbonate 
formed but has no inhibiting action on the enzyme [Harden, 1913 ; 
Neuberg and Rosenthal, 1913]. 

As already mentioned the action is exerted on a-ketonic acids 
as a class and proceeds with great readiness with oxalacetic acid, 
COOH -CH 2 CO COOH, all the three forms of which are decom- 
posed, with a-ketoglutaric acid, and with a-ketobutyric acid. Hy- 
droxypyruvic acid CH 2 (OH) * CO COOH is slowly decomposed 
yielding glycolaldehyde, CH 2 (OH) CHO, and this condenses to a 
sugar [Neuberg and Kerb, 1912,3; 1913, i]. Positive results have 
also been obtained with diketobutyric, phenylpyruvic, p-hydroxyphenyl- 
pyruvic, phenylglyoxylic and acetonedicarboxylic acids [Neuberg and 
Karczag, 1911, 5]. 

Relation of Carboxylase to Alcoholic Fermentation. 

With regard to the relation of carboxylase to the process of alcoholic 
fermentation, nothing definite is yet known. As Neuberg points out 
[see Neuberg and Kerb, 1913, i] the universal presence of the enzyme in 
yeasts capable of producing alcoholic fermentation, and the extreme 
readiness with which the fermentation of pyruvic acid takes place create a 



CARBOXYLASE 83 

strong presumption that the decomposition of pyruvic acid actually forms 
a stage in the process of the alcoholic fermentation of the sugars. On 
the other hand Ehrlich's alcoholic fermentation of the amino-acids 
(p. 87) provides another function for carboxylase that of decomposing 
the a-ketonic acids produced by the deaminisation of the amino-acids. 
It must be remembered in this connection that carboxylase is not 
specific in its action, but catalyses the decomposition not only of 
pyruvic acid but also of a large number of other a-ketonic acids, in- 
cluding many of those which correspond to the amino-acids of proteins 
and are doubtless formed in the characteristic decomposition of these 
amino-acids by yeast. Carboxylase undoubtedly effects one stage in 
the production of alcohols from amino-acids, whether it is also the 
agent by which one stage in the alcoholic fermentation of sugar is 
brought about still remains to be proved. 

A comparison of the conditions of action of carboxylase and 
zymase has revealed several interesting points of difference. Neuberg 
and Rosenthal [1913] have observed that the fermentation of pyruvic 
acid by maceration extract commences much more rapidly than that 
of glucose and interpret this to mean that in the fermentation of glucose 
a long preliminary process occurs before sufficient pyruvic acid has been 
produced to yield a perceptible amount of carbon dioxide. The long de- 
lay (3 hours) which they sometimes observed in the action of macera- 
tion juice on glucose is however by no means invariable (see p. 46), 
but in any case indicates that the sugar fermentation can be affected 
by conditions which are without influence on the pyruvic fermentation. 
A similar conclusion is to be drawn from the fact that the pyruvic acid 
fermentation is less affected by antiseptics than the glucose fermenta- 
tion [Neuberg and Karczag, 1911, 4; Neuberg and Rosenthal, 1913], 
chloroform sufficient to stop the glucose fermentation brought about 
by yeast or dried yeast being usually without effect on the fermenta- 
tion of the pyruvates either alone or in presence of boric or arsenious 
acid. A more important difference is that carboxylase decomposes 
pyruvic acid in the absence of the co-enzyme which is necessary for 
the fermentation of glucose [Harden, 1913 ; Neuberg and Rosenthal, 
I 9 I 3] This can be demonstrated experimentally by washing dried 
yeast or zymin with water (see p. 63) until it is no longer capable of 
decomposing glucose (Harden), or by allowing maceration extract to 
autolyse or dialyse until it is free from co-enzyme (Neuberg and 
Rosenthal). The zymase of maceration extract is moreover inacti- 
vated in 10 minutes at 50-51, whereas after this treatment the car- 
boxylase is still active. 

6 * 



84 ALCOHOLIC FERMENTATION 

The only conclusion that can be legitimately drawn from these 
highly interesting facts is that if the decomposition of pyruvic acid 
actually be a stage in the alcoholic fermentation of glucose the soluble 
co-enzyme is required for some change precedent to this, so that in its 
absence the production of pyruvic acid cannot be effected. 



CHAPTER VII. 

THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION. 

WHEN pure yeast is allowed to develop in a solution of sugar contain- 
ing a suitable nitrogenous diet and the proper mineral salts, the liquid 
at the close of the fermentation contains not only alcohol and some 
carbon dioxide but also a considerable number of other substances, 
some arising from the carbonaceous and others from the nitrogenous 
metabolism of the cell. Prominent among the non-nitrogenous sub- 
stances which are thus found in fermented sugar solutions are fusel oil, 
succinic acid, glycerol, acetic acid, aldehyde, formic acid, esters, and 
traces of many other aldehydes and acids. In addition to these sub- 
stances which are found in the liquid, there are also the carbonaceous 
constituents of the newly formed cells of the organism, comprising the 
material of the cell walls, yeast gum, glycogen, complex organic phos- 
phates, as well as other substances. 

The attention of chemists has been directed to these compounds 
since Pasteur first emphasised their importance as essential products 
of the alcoholic fermentation of sugar, and his example was generally 
followed in attributing their origin to the sugar. 

The study of cell-free fermentation by means of yeast-juice or 
zymin has, however, revealed the facts that certain of these substances 
are not formed in the absence of living cells, and that their origin is to 
be sought in the metabolic processes which accompany the life of the 
cell. Their source, moreover, has been traced not to the sugar but to 
the amino-acids, formed by the hydrolysis of the proteins, which occur 
in all such liquids as beer wort, grape juice, etc., which are usually 
submitted to alcoholic fermentation. This has so far been proved with 
certainty for the fusel oil and succinic acid, and rendered highly prob- 
able for all the various aldehydes and acids of which traces have been 
detected. 

Fusel Oil. 

All forms of alcohol prepared by fermentation contain a fraction of 
high boiling-point, which is termed fusel oil, and amounts to about 

85 



86 ALCOHOLIC FERMENTATION 

OT to 07 per cent, of the crude spirit obtained by distillation. This 
material is not an individual substance, but consists of a mixture of 
very varied compounds, all occurring in small amount relatively to the 
ethyl alcohol from which they have been separated. The chief con- 
stituents of the mixture are the two amyl alcohols, isoamyl alcohol, 

(CH 3 ) 2 .CH.CH 2 .CH 2 .OH, 
and dT-amyl alcohol, 

CH 3 .CH(C 2 H 5 ).CH 2 .OH, 

which contains an asymmetric carbon atom and is optically active. In 
addition to these, much smaller amounts of propyl alcohol and isobutyl 
alcohol are present, together with traces of fatty acids, aldehydes, and 
other substances. 

The origin of these purely non-nitrogenous compounds was usually 
sought in the sugar of the liquid fermented, from which they were 
thought to be formed by the yeast itself or by the agency of bacteria 
[Emmerling, 1904, 1905 ; Pringsheim, 1905, 1907, 1908, 1909], 
whilst others traced their formation to the direct reduction of fatty 
acids. Felix Ehrlich has, however, conclusively shown in a series of 
masterly researches that the alcohols, and probably also the aldehydes, 
contained in fusel oil are in reality derived from the amino-acids 
which are formed by the hydrolysis of the proteins. 

The close relationship between the composition of leucine, 

(CH 3 ) 2 .CH.CH 2 .CH(NH 2 ).COOH, 
and isoamyl alcohol, 

(CH 3 ) 2 .CH.CH 2 .CH 2 .OH, 

had previously led to the surmise that a genetic relation might exist 
between these substances, but the idea had not been experimentally 
confirmed. In 1903 Ehrlich discovered [1903 ; 1904, I, 2; 1907, 2; 
1908; Ehrlich and Wendel, 1908, 2] that proteins also yield on 
hydrolysis an isomeride of leucine known as isoleucine, which has the 
constitution 

CH 3 .CH(C 2 H 5 ).CH(NH 2 ).COOH, 

and therefore stands to <f-amyl alcohol, 

CH 3 .CH(C 2 H 5 ).CH 2 .OH, 

in precisely the same relation as leucine to isoamyl alcohol. This sug- 
gestive fact at once directed his attention to the problem of the origin 
of the amyl alcohols in alcoholic fermentation. Using a pure culture 
of yeast, and thus excluding the participation of bacteria in the change, 
he found that leucine readily yielded isoamyl alcohol, and isoleucine 
alcohol when these amino-acids were added in the pure state 



THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION 87 

to a solution of sugar and treated with a considerable proportion of yeast 
[1905 ; 1906, 2, 3 ; 1907, I, 3]. The chemical reactions involved are 
simple ones and are represented by the following equations : 



(i)(CH 3 ) 2 . CH . CH 2 . CH(NH a ) . COOH + H 2 O = (CH 3 ) 2 . CH.CH 2 . CH 2 . 

Leucine Isoamyl alcohol 

(2) CH 3 . CH(C 2 H 5 ) . CH(NH 2 ) . COOH + H 2 O= CH 3 . CH(C 2 H 6 ) . CH 2 . OH + CO 2 + NH 8 

Isoleucine d-Amyl alcohol 

The experiments by which these important changes were demon- 
strated were of a very simple and convincing character [Ehrlich, 1907, 
i]. Two hundred grams of sugar and 3 to 10 grams of the nitro- 
genous substance to be examined were dissolved in 2 to 2-5 litres of 
tap water in a 3 to 4 litre flask, the liquid was sterilised by being 
boiled for several hours, and after cooling 40 to 60 grams of fresh 
yeast were added and the flask allowed to stand at room temperature 
until the whole of the sugar had been decomposed by fermentation. 
In the earlier experiments the amyl alcohols were isolated and ident- 
ified by conversion into the corresponding valerianic acids, but as a 
rule the fusel oil as a whole was quantitatively estimated in the filtrate 
by the Rose-Herzfeld method [Lunge, 1905, p. 571]. 

The following are typical results. (i) An experiment carried 
out as above without any addition of leucine gave 97*32 grams of 
alcohol containing 0*40 per cent, of fusel oil. (2) When 6 grams 
of synthetic, optically inactive leucine were added, 97*26 grams of 
alcohol were obtained, containing 2'ii per cent, of fusel oil, which 
was also optically inactive; 2*5 grams of leucine were recovered, so 
that 87 per cent, of the theoretical yield of isoamyl alcohol was ob- 
tained from the 3-5 grams of leucine decomposed. (3) In the pre- 
sence of 2 -5 grams of af-isoleucine (prepared from molasses residues), 
200 grams of sugar gave 93*99 grams of alcohol, containing 1-44 per 
cent of fusel oil, which was laevo-rotatory. This corresponds with 80 
per cent, of the theoretical yield of ^-amyl alcohol from the isoleucine 
added 

This change, which Ehrlich has termed the alcoholic fermentation 
of the amino-acids, although brought about by living yeast, does not 
appear to occur at all when zymin [Ehrlich, 1906,4; Pringsheim, 
1906] or yeast-juice [Buchner and Meisenheimer, 1906] is substituted 
for the intact organism, nor is it effected even by living yeast in the 
absence of a fermentable sugar [Ehrlich, 1907, i]. The reaction appears 
indeed to be intimately connected with the nitrogenous metabolism of 
the cell, and the whole of the ammonia produced is at once assimilated 
and does not appear in the fermented liquid. Other amino-acids 



88 ALCOHOLIC FERMENTATION 

undergo a corresponding change, and the reaction appears to be a 
general one. Thus tyrosine, OH . C 6 H 4 . CH 2 . CH(NH 2 ) . COOH, 
yields p-hydroxyphenylethyl alcohol, or tyrosol [Ehrlich, 1911,1; Ehrlich 
and Pistschimucka, 1912, 2], OH . C 6 H 4 . CH 2 . CH 2 OH, a substance of 
intensely bitter taste, which was first prepared in this way and is pro- 
bably one of the most important factors in determining the flavour of 
beers, etc. Phenylalanine, C 6 H 5 . CH 2 . CH(NH 2 ) . COOH, in a 
similar way yields phenylethyl alcohol, C 6 H 5 . CH 2 . CH 2 OH, one of 
the constituents of oil of roses, whilst tryptophane, 

HNx' 6 4 ^>C.CH a .CH(NH 2 ).COOH, 
\ CH r 

yields tryptophol, 

HN / C H < 

which was also first prepared in this way [Ehrlich, 1912] and has a 
very faintly bitter, somewhat biting taste. 

The extent to which the amino-acids of a medium in which yeast 
is producing fermentation are decomposed in this sense depends on 
the amount of the available nitrogen and on the form in which it is 
present. Thus the addition of ammonium carbonate to a mixture of 
yeast and sugar was found to lower the production of fusel oil from 
07 to 0*33 per cent, of the alcohol produced. The addition of leucine 
alone raised the percentage from 07 to 278, but the addition of both 
leucine and ammonium carbonate resulted in the formation of only 
078 per cent, of fusel oil The production of fusel oil therefore and 
the character of the constituents of the fusel oil alike depend on the 
composition of the medium in which fermentation occurs. This affords 
a ready explanation of the fact that molasses, which contains almost 
equal amounts of leucine and isoleucine, yields a fusel oil also contain- 
ing approximately equal amounts of isoamyl alcohol and dT-amyl alcohol 
[Marckwald, 1902], whilst corn and potatoes, in which leucine 
preponderates over isoleucine, yield fusel oils containing a relatively 
large amount of the inactive alcohol. The subject is, in fact, one of 
great interest to the technologist, for as Ehrlich points out " the great 
variety of the bouquets of wine and aromas of brandy, cognac, arrak, 
rum, etc., may be very simply referred to the manifold variety of the 
proteins of the raw materials (grapes, corn, rice, sugar cane, etc.) from 
which they are derived ". 

Yeast can also form fusel oil at the expense of its own protein, 
but this only occurs to any considerable extent when the external 



THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION 89 

supply of nitrogen is insufficient. Under these circumstances the 
ammo-acids formed by autolysis may be decomposed and their 
nitrogen employed over again for the construction of the protein of 
the cell. 

The yield is also influenced by the condition of the yeast employed 
with regard to nitrogen, a yeast poor in nitrogen being more efficacious 
in decomposing amino-acids than one which is already well supplied 
with nitrogenous materials. The nature of the carbonaceous nutriment 
and finally the species of yeast are also of great importance [see 
Ehrlich, 1911, 2 ; Ehrlich and Jacobsen, 1911], 

A very important characteristic of the action of yeast on the 
amino-acids is that the two stereo-isomerides of these optically active 
compounds are fermented at different rates. When inactive, racemic 
leucine is treated with yeast and sugar, the naturally occurring com- 
ponent, the /-leucine, is more rapidly attacked, so that if the experi- 
ment be interrupted at the proper moment the other component, the 
^/-leucine, alone is present and may be isolated in the pure state. In an 
actual experiment 3-8 grams of this component were obtained in the 
pure state from 10 grams of ^//-leucine [Ehrlich, 1906, i], so that the 
whole of the /-leucine (5 grams) had been decomposed but only 1*2 
grams of the ^/-leucine. This mode of action has been found to be 
characteristic of the alcoholic fermentation of the amino-acids by yeast. 
In all the instances so far observed, both components of the inactive 
amino-acid are attacked, but usually the naturally occurring isomeride 
is the more rapidly decomposed, although in the case of /3-aminobutyric 
acid both components disappear at the same rate [Ehrlich and Wen- 
del, 1908, i]. This reaction therefore must be classed along with the 
action of moulds on hydroxy-acids [McKenzie and Harden, 1903], 
and the action of lipase on inactive esters [Dakin, 1903, 1905], in 
which both isomerides are attacked but at unequal rates, and differs 
sharply from the action of yeast itself on sugars [Fischer and Thier- 
felder, 1894], and of emulsin, maltase, etc., which only act on one isomer- 
ide and leave the other entirely untouched [see Bayliss, 1914, pp. 55, 
77, 117]- 

Succinic Acid. 

The origin of the succinic acid formed in fermentation has also 
been traced by Ehrlich [1909] to the alcoholic fermentation of the 
amino-acids. It was shown by Buchner and by Kunz [1906] that 
succinic acid like fusel oil is not formed during fermentation by yeast- 
juice or zymin, and, in the light of Ehrlich's work on fusel oil, several 



90 ALCOHOLIC FERMENTATION 

modes of formation appeared possible for this substance [Ehrlich, 1906, 
3]. The dibasic amino-acids might, for example, undergo simple 
reduction, the NH 2 group being removed as ammonia and replaced by 
hydrogen. Aspartic acid would thus pass into succinic acid : 

COOH-CH 2 -CH(NH 2 ).COOH + 2 H = 
COOH.CH 2 .CH 2 .COOH + NH 3 . 

This change can be effected in the laboratory only by heating with 
hydriodic acid. Biologically it has been observed [E. and H. Sal- 
kowski, 1879] when aspartic acid is submitted to the action of 
putrefactive bacteria, and almost quantitatively when Bacillus coli com- 
munis is cultivated in a mixture of aspartic acid and glucose [Harden, 
1901]. In this case a well-defined source of hydrogen exists in the 
glucose, which when acted on by this bacillus yields a large volume 
of gaseous hydrogen, which is not evolved in the presence of aspartic 
acid. Some such source is also available in the case of yeast, although 
it cannot be chemically defined, for this organism is known to produce 
many reducing actions, which are usually ascribed to the presence of 
reducing ferments or reductases in the cell. 

A similar action would convert glutamic acid, 

COOH . CH a . CH 2 . CH(NH) 2 . COOH, 
into glutaric acid, 

COOH . CH 2 . CH 2 . CH 2 . COOH, 

which also is found among the products of fermentation, whilst the 
monamino-acids would pass into the simple fatty acids. 

On submitting these ideas to the test of experiment, however, 
Erhlich found that the addition of aspartic acid did not in any way in- 
crease the yield of succinic acid, and that of all the amino-acids which 
were tried only glutamic acid, COOH . CH 2 . CH 2 . CH(NH 2 ) . COOH, 
produced a definite increase in the amount of this substance. Further 
experiments showed that glutamic acid was actually the source of the 
succinic acid, the relations being quite similar to those which exist for 
the production of fusel oil. 

Succinic acid is formed whenever sugar is fermented by yeast, even 
in the absence of added nitrogenous matter, and amounts to O'2 to O'6 
per cent, of the weight of the sugar decomposed, its origin in this case 
being the glutamic acid formed by the autolysis of the yeast protein. 
When some other source of nitrogen is present, such as asparagine or 
an ammonium salt, the amount falls to 0*05 to OT. If glutamic acid 
be added it rises to about I to i'5 per cent, but falls again to about 
0*05 to 0*1 when other sources of nitrogen, such as asparagine or am- 
m,onium salts, are simultaneously available, either in the presence or 



THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION 91 

absence of added glutamic acid. As in the case of fusel oil, the pro- 
duction does not occur in the absence of sugar, and is not effected by 
yeast-juice or zymin. 

The chemical reaction involved in the production of succinic acid 
differs to some extent from that by which fusel oil is formed, inasmuch 
as an oxidation is involved : 

COOH . CH 2 . CH . CH(NH 2 ) . COOH + zO = 
COOH . CH 2 . CH 2 . COOH + NH 3 + CO 2 . 

From analogy with the production of amyl alcohol from leucine, 
glutamic acid would be expected to yield 7-hydroxybutyric acid : 
COOH . CH 2 . CH 2 . CH(NH 2 ) . COOH + H 2 O = NH 3 -f CO 2 + COOH . CH 2 . CH 2 . CH 2 . OH. 

As a matter of fact this substance cannot be detected among the 
products of fermentation, but succinic acid as already explained is 
formed. This acid might, however, possibly be formed by the oxidation 
of the 7-hydroxybutyric acid : 

COOH . CH 2 . CH 2 . CH 2 . OH . + 2 O = COOH . CH 2 . CH 2 . COOH + H 2 O, 
although this change is on biological grounds improbable. 

The conversion of the group CH(NH 2 ) into the terminal 
CH2.OH in fusel oil, or COOH in succinic acid, may possibly be 
effected in several different ways, the most probable of which are the 
following : 

I. Direct elimination of carbon dioxide, followed by hydrolysis of 
the resulting amine : 

(1) R. CH(NH 2 ) . COOH = R . CH 2 . NH 2 + CO 2 . 

(2) R.CH 2 .NH 2 +H 2 O = R.CH 



The reaction (i) is actually effected by many bacteria and has been 
employed for the preparation of bases from amino-acids [cf. Barger, 
1914, p. 7], although there is no direct evidence that it can be brought 
about by yeast. On the other hand reaction (2) has actually been ob- 
served with some yeasts. Thus it has been found [Ehrlich and Pist- 
schimuka, 1912, i] that many " wild " yeasts produce this change with 
great readiness in presence of sugar, glycerol or ethyl alcohol as 
sources of carbon and grow well in media in which amines, such as p- 
hydroxyphenylethylamine or iso-amylamine, form the only source of 
nitrogen. Willia anomala (Hansen), a yeast which forms surface 
growths, succeeds admirably under these conditions, whereas culture 
yeasts are much less active in this way, although they produce a cer- 
tain amount of change. It is therefore possible that this mode of de- 
composition plays some part in the production of fusel oil, but in the 
case of culture yeasts it is entirely subordinated to the mode next to 
be discussed. 



92 ALCOHOLIC FERMENTATION 

II. Oxidative removal of the -NH 2 group with formation of an 
a-ketonic acid : 

(i) R . CH(NH 2 ) . COOH + O= R . CO . COOH + NH 3 

followed by the decomposition of the ketonic acid into carbon dioxide 
and an aldehyde and the subsequent reduction or oxidation of the 

aldehyde : 

(2) R.CO.COOH=R.CHO + CO 2 . 

(3) (a) R.CHO-f 2H = R.CH 2 OH. 
(b) R.CHO + = R.COOH. 

The evidence for the occurrence of reaction (i) is supplied by the 
experiments of Neubauer and Fromherz [191 ij Having previously 
found that amino-acids undergo a change of this kind in the animal 
body, Neubauer investigated their behaviour towards yeast. Taking 
<//-phenylaminoacetic acid, C 6 H 5 . CH(NH 2 ) . COOH, it was found 
that the changes produced were essentially the same as in the animal 
body. The /-component of the acid was partly acetylated and partly 
unchanged, whereas the ^-component of the acid yielded benzyl alcohol, 
C 6 H 5 . CH 2 . OH, phenylglyoxylic acid, C 6 H 5 . CO . COOH, and the 
hydroxy-acid C 6 H 5 . CH(OH) . COOH. Since however this hydroxy- 
acid was produced in the /-form it probably arose by the asymmetric 
reduction of phenylglyoxylic acid, a reaction which can be effected by 
yeast as was also found to be the case in the animal body [see Dakin, 
1 91 2, pp. 5 2, 78]. Moreover it was shown that when the effects of yeast 
on a ketonic acid and the corresponding hydroxy-acid were compared, 
the alcohol was formed in much better yield from the ketonic acid (70 
per cent.) than from the hydroxy-acid (3-4 per cent), the actual 
example being the production of tyrosol (p-hydroxyphenyl ethyl 
alcohol),OH . C 6 H 4 . CH 2 . C H 2 OH, from p-hydroxyphenylpyruvic acid, 
OH . C 6 H 4 . CH 2 . CO . COOH, and p-hydroxyphenyl-lactic acid, 

OH . C 6 H 4 . CH 2 . CH(OH) . COOH 

respectively. 

Neubauer by these experiments established two extremely import- 
ant points, i. That the amino-acids actually yield the corresponding 
a-ketonic acids when treated with yeast and sugar solution. 2. That 
the a-ketonic acids under similar conditions give the alcohol contain- 
ing one carbon atom less in good yield, whereas the corresponding 
hydroxy-acids only give an extremely small amount of these alcohols. 

It is therefore probable that at an early stage in the decomposi- 
tion of the amino-acids by yeast a ketonic acid is produced, which 
then undergoes further change. 

The source of the oxygen required for this reaction and the mechan- 
ism of oxidation have not yet been definitely ascertained. It is possible 



THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION 93 

/OH 

that hydrated imino-acids of the type R . C COOH are first formed 



[Knoop, 1910], but these have not as yet been isolated. 

The spontaneous production of ketonic aldehydes from amino-acids 
and from hydroxy-acids in aqueous solution, which has been demon- 
strated by Dakin and Dudley [1913], points however to the possibility 
that the ketonic acid may be a secondary product derived from the 
corresponding ketonic aldehyde [see also Dakin, 1908 ; Neuberg, 1908, 
1909]. This itself may either arise directly from the amino-acid or 
from a previously formed hydroxy-acid, the latter alternative being, 
however, improbable in view of the small yield of alcohol obtained from 
hydroxy-acids by the action of yeast in the experiments of Neubauer 
and Fromherz. 

R . CH(NH a ) . COOH - R . CH(OH) . COOH 

^ // 

R.CO.CHO 

| + oxygen 
R. CO. COOH 

(2) Whatever be the exact mode by which the ketonic acid is 
formed, it appears most probable that a compound of this nature forms 
the starting-point for the next stage in the production of the alcohols. 
The researches of Neuberg, which have already been discussed on p. 
81, have revealed a mechanism in yeast the enzyme carboxylase 
by which these ^-ketonic acids are rapidly broken up into an aldehyde 
and carbon dioxide: 

R . CO . COOH = R . CHO + CO 2 

and it can scarcely be doubted that this is the actual course of the 
reaction. 

(3) The final conversion of the aldehyde into the corresponding 
alcohol is also a change which it has been proved can be effected by 
yeast [Neuberg and Rosen thai, 1913] probably by the aid of the 
reductase which is one of the weapons in its armoury of enzymes. 

Yeast is capable of producing many vigorous reducing actions and 
rapidly reduces methylene blue and sodium selenite. It is in all 
probability due to a reaction of this kind that the iso-amylaldehyde 
and isovaleraldehyde were reduced to the alcohols in Neuberg and 
Steenbock's experiments [1913, 1914], and that considerable quantities 
of ethyl alcohol are formed in the sugar free fermentation of pyruvic 
acid [Neuberg and Kerb, 1913, i] (see later p. no for a discussion of 
this question). 

A further possibility exists that in some cases the aldehyde may 




94 ALCOHOLIC FERMENTATION 

be simultaneously oxidised and reduced or the molecule of one 
aldehyde reduced and that of another oxidised with production of 
the corresponding acid and alcohol by an " aldehydo-mutase," similar 
to that which has been observed by Parnas [1910] in many animal 
tissues. Finally the aldehyde may simply be converted into the 
corresponding acid by oxidation as appears to take place in the for- 
mation of succinic acid. 

The intermediate production of an aldehyde would thus be consist- 
ent both with the production of alcohols and acids from amino-acids. 

Fusel oil would be formed by the reduction of the aldehydes aris- 
ing from the simple monobasic amino-acids, succinic acid would be 
produced by oxidation of the aldehyde derived from the dibasic glu- 
tamic acid. 

In favour of this view is to be adduced the fact that aldehydes 
such as isobutyraldehyde and valeraldehyde have been found in crude 
spirit, whilst acetaldehyde is a regular product of alcoholic fermentation 
[see Ashdown and Hewitt, 1910]. Benzaldehyde, moreover, has 
been actually detected as a product of the alcoholic fermentation of 
phenylaminoacetic acid, C 6 H 5 . CH(NH 2 ). COOH [Ehrlich, 1907, i]. 
Further, the aldehydes so produced would readily pass by oxidation 
into the corresponding fatty acids, small quantities of which are in- 
variably produced in fermentation. 

This view of the nature of the alcoholic fermentation of the amino- 
acids is undoubtedly to be preferred to that previously suggested by 
Ehrlich [1906, 3] according to which a hydroxy-acid is first formed 
and then either directly decomposed into an alcohol and carbon dioxide 
or into an aldehyde and formic acid, the aldehyde being reduced and 
the formic acid destroyed (see p. 115). 

R . CH(NH a ) . COOH -> R . CH(OH) . COOH 

iS ^ 

R . CH 2 OH + C0 2 R . CHO + H CO 2 H 

R-CH 2 OH 

The most probable course of the decomposition by which isoamyl 
alcohol and succinic acid are produced from leucine and glutamic acid 
respectively is therefore the following : 

(a) Isoamyt Alcohol. 

(1) (CH 8 ) a . CH . CH 2 . CH(NH 2 ) . COOH (3) (CH 3 ) 2 . CH . CH 2 . CHO + CO a 

Leucine' Isovaleraldehyde 

(2) (CH 3 ) 2 . CH . CH ? . CO . COOH (4) (CH 8 ) 2 . CH . CH 2 . CH 2 OH 

a-Ketoisovalerianic acid Isoamyl alcohol 

(b) Succinic Acid. 

(1) COOH . CH 2 . CH 2 . CH(NH 2 ) . COOH (3) COOH . CH 3 . CH 2 . CHO + CO 2 

Glutamic acid Succinic semialdehyde 

(2) COOH . CH 2 CH 2 . CO . COOH (4) COOH . CH 2 . CH 2 . COOH 

a-Keto-glutaric acid Succinic acid 



THE BY-PRODUCTS OF ALCOHOLIC FERMENTATION 95 

Glycerol. 

Of the three chief by-products of alcoholic fermentation, only 
glycerol remains at present referable directly to the sugar. This 
substance, as shown by the careful experiments of Buchner and 
Meisenheimer [1906], is formed by the action both of yeast-juice 
and zymin to the extent of 3*8 per cent, of the sugar decomposed, and 
no other source for its production has so far been experimentally de- 
monstrated. If it be true that during the decomposition of sugar 
into alcohol and carbon dioxide, substances containing three carbon 
atoms are formed as intermediate compounds (see p. 100), it is obvious 
that these might by reduction be converted into glycerol which would 
thus be a true by-product of the alcoholic fermentation of sugar. [See 
Oppenheimer, 1914, 2.] It has, however, been suggested that it may 
in reality be a product of decomposition of lipoid substances or of 
the nuclein of the cell (Ehrlich). 

The effect of Ehrlich's work has been clearly to distinguish the 
chemical changes involved in the production of fusel oil and succinic 
acid from those concerned in the decomposition of sugar into alcohol 
and carbon dioxide, and to bring to light a most important series of 
reactions by means of which the yeast-cell is able to supply itself 
with nitrogen, one of the indispensable conditions of life. 



CHAPTER VIII. 

THE CHEMICAL CHANGES INVOLVED IN FERMENTATION. 

IT has long been the opinion of chemists that the remarkable and 
almost quantitative conversion of sugar into alcohol and carbon dioxide 
during the process of fermentation is most probably the result of a 
series of reactions, during which various intermediate products are 
momentarily formed and then used up in the succeeding stage of the 
process. No very good ground can be adduced for this belief except 
the contrast between the chemical complexity of the sugar molecule 
and the comparative simplicity of the constitution of the products. 
Many attempts have, however, been made to obtain evidence of such a 
series of reactions, and numerous suggestions have been made of prob- 
able directions in which such changes might proceed. In making 
these suggestions, investigators have been guided mainly by the changes 
which are produced in the hexoses by reagents of known composition. 
The fermentable hexoses, glucose, fructose, mannose, and galactose, 
appear to be relatively stable in the presence of dilute acids at the 
ordinary temperature, and are only slowly decomposed at 100, more 
rapidly by concentrated acids, with formation of ketonic acids, such 
as levulinic acid, and of coloured substances of complex and unknown 
constitution. 

In the presence of alkalis, on the other hand, the sugar molecule is 
extremely susceptible of change. In the first place, as was discovered 
by Lobry de Bruyn [1895 ; Bruyn and Ekenstein, 1895 >' ^96; 1897, 
I, 2, 3, 4], each of the three hexoses, glucose, fructose, and mannose 
is converted by dilute alkalis into an optically almost inactive mixture 
containing all three, and probably ultimately of the same composition 
whichever hexose is employed as the starting-point. 

This interesting phenomenon is most simply explained on the as- 
sumption that in the aqueous solution of any one of these hexoses, 
along with the molecules of the hexose itself, there exists a small pro- 
portion of those of an enolic form which is common to all the three 
hexoses, as illustrated by the following formulae, the aldehyde formulae 

96 



CHEMICAL CHANGES INVOLVED IN FERMENTATION 97 



being employed instead of the 7-oxide formulae for the sake of 
simplicity : 

CHO CHO CH a (OH) CH(OH) 

HCOH HOCH CO COH 

HOCH HOCH HOCH HOCH 

HCOH HCOH HCOH HCOH 

HCOH HCOH HCOH HCOH 

fill 
CH 2 (OH) CH 2 (OH) CH 2 (OH) CH 2 (OH) 

Glucose. Mannose. Fructose. Enolic form. 

This enolic form is capable of giving rise to all three hexoses, and 
the change by which the enolic form is produced and converted into 
an equilibrium mixture of the three corresponding hexoses is catalyt- 
ically accelerated by alkalis, or rather by hydroxyl ions. In neutral 
solution the change is so slow that it has never been experimentally 
observed ; in the presence of decinormal caustic soda solution at 70 
the conversion is complete in three hours. Precisely similar effects are 
produced with galactose, which yields an equilibrium mixture contain- 
ing talose and tagatose, sugars which appear not to be fermentable. 

The continued action even of dilute alkaline solutions carries the 
change much further and brings about a complex decomposition 
which is much more rapidly effected by more concentrated alkalis 
and at higher temperatures. This change has been the subject of 
very numerous investigations [for an account of these see E. v. 
Lippmann, 1904, pp. 328, 713, 835], but for the present purpose the 
results recently obtained by Meisenheimer [1908] may be quoted as 
typical. Using normal solutions of caustic soda and concentrations 
of from 2 to 5 grams of hexose per 100 c.c., it was found that at 
air temperature in 27 to 139 days from 30 to 54 per cent, of the 
hexose was converted into inactive lactic acid, C 3 H 6 O 3 , from 0*5 to 
2 per cent, into formic acid, CH 2 O 2 , and about 40 per cent, into a 
complex mixture of hydroxy-acids, containing six and four carbon 
atoms in the molecule. Usually only about 74 to 90 per cent, of the 
sugar which had disappeared was accounted for, but in one case the 
products amounted to 97 per cent, of the sugar. About I per cent, 
of the sugar was probably converted into alcohol and carbon dioxide. 
No glycollic acid, oxalic acid, glycol, or glycerol was produced. 

The fact that alcohol is actually formed by the action of alkalis on 
sugar was established by Buchner and Meisenheimer [1905], who ob- 
tained small quantities of alcohol (i'8 to 2*8 grams from 3 kilos, of cane 
sugar) by acting on cane sugar with boiling concentrated caustic soda 

7 



98 ALCOHOLIC FERMENTATION 

solution. It is evident that under these conditions an extremely com- 
plex series of reactions occurs, but the formation of alcohol and carbon 
dioxide and of a large proportion of lactic acid deserves more particular 
attention. 

The direct formation of alcohol from sugar by the action of alkalis 
appears first to have been observed by Duclaux [1886], who ex- 
posed a solution of glucose and caustic potash to sunlight and obtained 
both alcohol and carbon dioxide. As much as 2 -6 per cent, of the sugar 
was converted into alcohol in a similar experiment made by Buchner and 
Meisenheimer [1904]. When the weaker alkalis, lime water or baryta 
water, were employed instead of caustic potash, however, no alcohol was 
formed, but 50 per cent, of the sugar was converted into inactive lactic 
acid [Duclaux, 1893, 1896]. Duclaux therefore regarded the alcohol 
and carbon dioxide as secondary products of the action of a com- 
paratively strong alkali on preformed lactic acid. Ethyl alcohol can, 
in fact, be produced from lactic acid both by the action of bacteria 
[Fitz, 1880] and of moulds [Maze, 1902], and also by chemical means. 
Thus Duclaux [1886] found that calcium lactate solution exposed to 
sunlight underwent decomposition, yielding alcohol and calcium car- 
bonate and acetate, whilst Hanriot [1885, 1886], by heating calcium 
lactate with slaked lime obtained a considerable quantity of a liquid 
which he regarded as ethyl alcohol, but which was shown by Buchner 
and Meisenheimer [1905] to be a mixture of ethyl alcohol with 
isopropyl alcohol. 

It appears, therefore, that inactive lactic acid can be quite readily 
obtained in large proportion from the sugars by the action of alkalis, 
whilst alcohol can only be prepared in comparatively small amount and 
probably only as a secondary product of the decomposition of lactic 
acid. 

The study of the action of alkalis on sugar has, however, yielded 
still further information as regards the mechanism of the reaction by 
which lactic acid is formed. A considerable body of evidence has 
accumulated, tending to show that some intermediate product of the 
nature of an aldehyde or ketone containing three carbon atoms is first 
formed. 

Thus Pinkus [1898] and subsequently Nef [1904, 1907], by act- 
ing on glucose with alkali in presence of phenylhydrazine ob- 
tained the osazone of methylglyoxal, CH 3 . CO . CHO. This osazone 
may be formed either from methylglyoxal itself, from acetol, 
CH 3 . CO . CH 2 . OH, or from lactic aldehyde, CH 3 . CH(OH) . CHO 
[Wohl, 1908]. Methylglyoxal itself may also be regarded as a second- 



CHEMICAL CHANGES INVOLVED IN FERMENTATION 99 

ary product derived from glyceraldehyde, CH 2 (OH). CH(OH). CHO, 
or dihydroxyacetone, CH 2 (OH) . CO . CH 2 (OH), by a process of intra- 
molecular dehydration, so that the osazone might also be derived 
indirectly from either of these compounds [see also Neuberg and 
Oertel, 1913]. Methylglyoxal itself readily passes into lactic acid 
when it is treated with alkalis, a molecule of water being taken up : 
CH 3 . CO . CHO + H 2 = CH 3 . CH(OH) .COOH. 

Further evidence in the same direction is afforded by the interesting 
discovery of Windaus and Knoop [1905], that glucose is converted by 
ammonia in presence of zinc hydroxide into methyliminoazole, 



CH 



.isr 



a substance which is a derivative of methylglyoxal. 

The idea suggested by Pinkus that acetol is the first product of 
the action of alkalis on sugar has been rendered very improbable by 
the experiments of Nef, and the prevailing view (Nef, Windaus and 
Knoop, Buchner and Meisenheimer) is that the first product is glycer- 
aldehyde, which then passes into methylglyoxal, and finally into lactic 
acid : 

(1) C 6 H 12 O 6 =2CH.,(OH) . CH(OH) . CHO. 

(2) CH 2 (OH) . CH(OH) . CHO = CH 3 . CO . CHO + H a O. 

(3) CH 3 . CO . CHO + H 2 O = CH 3 . CH(OH) . COOH. 

All these changes may occur at ordinary temperatures in the presence 
of a catalyst, and in so far resemble the processes of fermentation by 
yeasts and bacteria. 

The first attempt to suggest a scheme of chemical reactions by 
which the changes brought about by living organisms might be 
effected was made in 1870 by Baeyer [1870], who pointed out that 
these decompositions might be produced by the successive removal 
and re-addition of the elements of water. The result of this would 
be to cause an accumulation of oxygen atoms towards the centre of 
the chain of six carbon atoms, which, in accordance with general ex- 
perience, would render the chain more easily broken. Baeyer form- 
ulated the changes characteristic of the alcoholic and lactic fermenta- 
tions as follows, the intermediate stages being derived from the 
hydrated aldehyde formula of glucose by the successive removal and 
addition of the elements of water : 



7* 



ioo ALCOHOLIC FERMENTATION 



I. 


II. 


III. 


IV. 


V. 


CH 2 .OH 


CH 2 ... OH 


CH 3 


CH 3 


CH 3 


CH.OH 


COH . . H 


CH.OH 


CH(OH) 


CH 

XVxJTlo 


| 


| 


| 







CH.OH 


C. .OH. .H 


C(OH) 2 


o 


\co 


CH.OH 

CH.OH 

j 


COH . . . H 

COH...H 

1 


C(OH) 2 
C(OH) 2 


XO 

C:H(OH) 


,\J\J 

\ 

^co 


CH(OH) 2 


CH . . . (OH) 2 


CH 3 


CH 


1 2 



The immediate precursor of alcohol and carbon dioxide is here seen 
to be the anhydride of ethoxycarboxylic acid (V), whilst that of lactic 
acid is lactic anhydride (IV). (Baeyer does not appear, as recently 
stated by Meisenheimer [1907, p. 8], Wohl [1907, 2], and Buchner 
and Meisenheimer [1909] to have suggested that lactic acid was an 
intermediate product in alcoholic fermentation, but rather to have 
represented independently the course of the two different kinds of 
fermentation, the alcoholic and the lactic.) 

It was subsequently pointed out by Buchner and Meisenheimer 
[1904] that Baeyer's principle of oxygen accumulation might be applied 
in a different way, so that a ketonic acid would be produced, the 
decomposition of which, in a manner analogous to that of acetoacetic 
acid, would lead to the formation of two molecules of lactic acid, from 
which the final products alcohol and carbon dioxide might be directly 
derived, as shown in the following formulae : 

CO., 

CH 2 . OH 
CH 3 
CO 2 



CHO 


COOH 


COOH 


CH(OH) 


CH(OH) 


CH(OH) 


CH(OH) 


CH 2 


CH 3 


CH(OH) 


CO 


COOH 


CH(OH) 


CH(OH) 


CH(OH) 


CH 2 (OH) 


CH 3 


CH 3 



CH 2 . OH 
CH 3 

A scheme based on somewhat different principles has been propounded 
by Wohl [Lippmann, 1904, p. 1 891], and has been accepted by Buchner 
and Meisenheimer [1905] as more probable than that quoted above. 
Wohl and Oesterlin [1901] were able to trace experimentally the 
various stages of the conversion of tartaric acid (I) into oxalacetic 
acid (III), which can be carried out by reactions taking place at the 
ordinary temperature, and they found that the first stage consisted 
in the removal of the elements of water leaving an unsaturated 
hydroxy derivative (II) which in the second stage underwent intra- 
molecular change into the corresponding keto-compound (III): 



CHEMICAL CHANGES INVOLVED IN FERMENTATION 101 



COOH 




COOH 


COOH 


CH(OH) 


H 


C(OH) 


- co 


CH(OH) 


OH ~ 


II 


CH 2 


COOH 




CH 


COOH 



III. 

Oxalacetic acid. 



COOH 

I. II. 

Tartaric acid. 

This change differs in principle from that assumed by Baeyer, inas- 
much as the second stage is not effected by the re-addition of water, 
but by the keto-enol transformation, which is now usually ascribed to 
the migration of the hydrogen atom, although the same result can 
theoretically be arrived at by the addition and removal of the elements 
of water. The analogy of this process to what might be supposed to 
occur in the conversion of sugar into carbon dioxide and alcohol was 
pointed out by Wohl and Oesterlin, and subsequently Wohl developed 
a theoretical scheme of reactions by which the process of alcoholic 
fermentation could be represented. In the first place the elements of 
water are removed from the a and /3 carbon atoms of glucose (I) and 
the resulting enol (II) undergoes conversion into the corresponding 
ketone (III), which has the constitution of a condensation product of 
methylglyoxal and glyceraldehyde, and hence is readily resolved by 
hydrolysis into these compounds (IV). The glyceraldehyde passes by 
a similar series of changes (V, VI) into methylglyoxal, and this is 
then converted by addition of water into lactic acid (VII), a reaction 
which is common to all ketoaldehydes of this kind. Finally, the lactic 
acid is split up into alcohol and carbon dioxide (VIII) : 



CHO 


CHO 


CHO 




CH(OH) H 


C(OH) 


CO 




| - OH 
CH(OH) 


II 
CH 2 


CH 2 




CH(OH) 


CH(OH) 


CH(OH) 




CH(OH) 


CH(OH) 


CH(OH) 




CH 2 (OH) 


CH 2 (OH) 


CH 2 (OH) 




I. 


II. 


III. 




Glucose. 








Methylglyoxal 








CHO 




COOH 


C0 2 


1 




| 






CO +H 2 




CH(OH) 


CH 2 OH 


| 




I 


| 


CH 3 




CH 3 


CH 3 


CHO CHO 


CHO 


COOH 


CO 2 


CH(OH) H C(OH) ^=- 


CO + H 2 O 


CH(OH) 




CH 2 OH 


1 OH || 


1 


| 


1 


CH 2 (OH) CH 2 


CH 3 


CH 3 


CH 3 


IV. V. 


VI. 


VII. 


VIII. 


Glyceraldehyde. Methylglyoxal. 


Lactic 


Alcohol 






acid. 


and 








carbon 








dioxide. 



102 ALCOHOLIC FERMENTATION 

This scheme agrees well with the current ideas as to the formation 
of lactic acid from glucose under the influence of alkalis (p. 99). It 
postulates the formation as intermediate products of no less than three 
compounds containing a chain of three carbon atoms glyceraldehyde, 
methylglyoxal, and lactic acid. 

The Lactic Acid Theory of Alcoholic Fermentation. 

A practical interest was given to these various schemes by the fact 
that Buchner and Meisenheimer adduced experimental evidence in 
favour of the view that lactic acid is an intermediate product in the 
formation of alcohol and carbon dioxide from sugar by fermentation 
[1904, 1905, 1906* 1909]- 

These observers proved by a series of very careful analyses 
that yeast-juice frequently, but not invariably, contains small quantities 
of lactic acid, not exceeding O'2 per cent. When yeast-juice is in- 
cubated alone or with sugar the amount of lactic acid may either 
increase or decrease. Moreover, lactic acid added to the juice is some- 
times diminished and sometimes increased in quantity. On the whole 
it appears that the addition of a considerable quantity of sugar or of 
some lactic acid favours the disappearance of lactic acid. Juices of 
low fermenting power produce a diminution in the lactic acid present, 
those of high fermenting power an increase. 

In all cases the amounts of lactic acid either produced or destroyed 
are very small in relation to the volume of the yeast-juice employed. 

Throughout the whole series of experiments the greatest increase 
amounted to 0*47 per cent, on the juice employed, and the greatest de- 
crease to 0*3 per cent. [See also Oppenheimer, 1914, I.] Buchner and 
Meisenheimer at one time regarded these facts as strong evidence that 
lactic acid is an intermediate product of alcoholic fermentation. It was 
thought probable that the production of alcohol and carbon dioxide 
from sugar occurred in at least two stages and under the influence of 
two distinct enzymes. The first stage consisted in the conversion of 
sugar into lactic acid, and for the enzyme which brought about this 
decomposition was reserved the name zymase or yeast-zymase. The 
lactic acid was then broken down into alcohol and carbon dioxide by 
the second enzyme, lactacidase. 

This theory, which is quite in harmony with the current ideas as to 
the mode of decomposition of sugars by alkalis, and is also consistent 
with Wohl's scheme of reactions, is open to adverse criticism from 
several points of view. In the first place, it is noticeable that the total 
amount of lactic acid used up by the juice is extremely small, even 



CHEMICAL CHANGES INVOLVED IN FERMENTATION 103 

in the most favourable cases, relatively to the amount of the juice 
[Harden, 1905], and it may be added to the sugar- fermenting power 
of the juice. Moreover, as pointed out by Buchner and Meisenheimer 
themselves [1909], no proof is afforded that the lactic acid which 
disappears is converted into alcohol and carbon dioxide. It is not 
even certain, although doubtless probable, that the lactic acid which 
occurs or is produced in the juice is really derived from sugar. 

The most weighty criticism of the theory is that of Slator [1906, 
1907 ; 1908, I, 2], which is based on the consideration that if lactic 
acid be an intermediate product of alcoholic fermentation the reaction 
by which it is fermented must proceed at least as rapidly as that by 
which it is formed, in order to prevent accumulation of lactic acid. 
The fermentation of lactic acid by yeast should therefore proceed at 
least as rapidly as that of glucose. So far is that from being the case 
that it has been experimentally demonstrated that lactic acid is not 
fermented at all by living yeast. This conclusion was rendered ex- 
tremely probable by Slator, who showed that lactic acid, even in con- 
centrations insufficient to prevent the fermentation of glucose, is not 
fermented to any considerable extent. The final proof that lactic acid 
is neither formed nor fermented by pure yeast has been brought by 
Buchner and Meisenheimer in a series of very careful quantitative 
experiments carried out with a pure yeast and with strict precautions 
against bacterial contamination [1909, 1910]. 

At first sight this fact appears decisive against the validity of the 
lactic acid theory, and it is recognised as such by Buchner and Meisen- 
heimer. Wohl has, however, suggested that the non-fermentability of 
lactic acid by yeast is not really conclusive [1907, I ; see also Franzen 
and Steppuhn, 1912, i]. The production of lactic acid from glucose 
is attended by the evolution of a considerable amount of heat (22 
cal), and it is possible that at the moment of production the molecule 
of the acid is in a condition of activity corresponding with a much 
higher temperature than the average temperature of the fermenting 
liquid. Under these circumstances the molecule would be much more 
susceptible of chemical change than at a later period when temperature 
equilibrium had been attained. It has, however, been pointed out 
by Tafel [1907], that such a decomposition of the lactic acid would 
occur at the very instant of formation of the molecule, so that no ground 
remains even on this view for assuming the actual existence of lactic 
acid as a definite intermediate product. It has also been suggested 
by Luther [1907] that an unknown isomeride of lactic acid is formed 
as an intermediate product and fermented, and that traces of lactic 



104 ALCOHOLIC FERMENTATION 

acid are formed by a secondary reaction from this, but no satisfactory 
evidence for this view is forthcoming. There still remains a doubt 
as to whether the living yeast-cell is permeable to lactic acid, a fact 
which would of course afford a very simple explanation of the non- 
fermentability of the acid. Apart from this, however, it is difficult, in 
face of the evidence just quoted, to believe that lactic acid is in reality 
an intermediate product in alcoholic fermentation. 

Methylglyoxal, Dihydroxyacetone and Glyceraldehyde. 

As regards the fermentability by yeast of compounds containing 
three carbon atoms, which may possibly appear as intermediate pro- 
ducts in the transformation of sugar into carbon dioxide and alcohol, 
many experiments have been carried out, with somewhat uncertain 
results. Care has to be taken that the substance to be tested is not 
added in such quantity as to inhibit the fermenting power of the yeast 
or yeast-juice, and further that the conditions are such that the sub- 
stance in question, often of a very unstable nature, is not converted by 
some chemical change into a different fermentable compound. It is 
also possible that the substance to be tested may accelerate the rate of 
autofermentation in a similar manner to arsenates (pp. 80, 126) and 
many other substances. These are all points which have not up to the 
present received sufficient attention. In the case of living yeast the 
further question arises of the permeability of the cell. 

Methylglyoxal, CH 3 . CO . CHO, has been tested by Mayer [1907] 
and Wohl [1907, 2] with yeast, and by Buchner and Meisenheimer both 
with acetone-yeast [1906] and yeast-juice [1910], in every case with 
negative results, but it may be noted that the concentration employed 
in the last mentioned of these experiments was such as considerably 
to diminish the autofermentation of the juice. 

Glyceraldehyde, CH 2 (OH) . CH (OH) . CHO, was also tested with 
yeast with negative results by Wohl [1898] and by Emmerling [1899], 
who employed a number of different yeasts. The same negative result 
attended the experiments of Piloty [1897] and Emmerling [1899] with 
pure dihydroxyacetone. Fischer and Tafel [1888, 1889], however, 
had previously found that glycerose, a mixture of glyceraldehyde and 
dihydroxyacetone prepared by the oxidation of glycerol, was readily 
fermented by yeast, agreeing in this respect with the still older obser- 
vations of Van Deen and of Grimaux. The reason for this diversity 
of result has not been definitely ascertained, but it has been supposed 
by Emmerling to lie in the formation of some fermentable sugar from 



CHEMICAL CHANGES INVOLVED IN FERMENTATION 105 

glycerose when the latter is subjected to too high a temperature during 
its preparation. 

On the other hand, Bertrand [1904] succeeded in fermenting pure 
dihydroxyacetone by treating a solution of I gram in 30 c.c. of liquid 
with a small quantity of yeast for ten days at 30, the best result being 
a fermentation of 2 5 per cent, of the substance taken. Moreover, Boysen- 
Jensen [1908, 1910, 1914] states that he has also observed both the 
formation from glucose and the fermentation of this substance by 
living yeast, but the amounts of alcohol and carbon dioxide produced 
were so minute and the evidence for the production of dihydroxy- 
acetone so inconclusive that the experiments cannot be regarded as in any 
way decisive [see Chick, 1912 ; Euler and Fodor, 1911 ; KarauschanofT, 
1911 ; Buchner and Meisenheimer, 1912]. A careful investigation by 
Buchner [1910] and Buchner and Meisenheimer [1910] has led them to 
the conclusion that both glyceraldehyde and dihydroxyacetone are 
fermentable. Glyceraldehyde exerts a powerful inhibiting action both 
on yeast and yeast-juice, and was only found to give rise to a very 
limited amount of carbon dioxide, quantities of cri5 to 0*025 gram 
being treated with I gram of yeast or 5 c.c. of yeast-juice and a pro- 
duction of 4 to 12 c.c. of carbon dioxide being attained. 

WhenO'i gram of dihydroxyacetone in 5 c.c. of water was brought 
in contact with I gram of living yeast, about half was fermented, 
17 c.c. of carbon dioxide (at 20 and 600 mm.) being evolved in excess 
of the autofermentation of the yeast (13 c.c.). A much greater effect 
was obtained by the aid of yeast-juice, and the remarkable observation 
was made that whilst yeast-juice alone produced comparatively little 
action a mixture of yeast-juice and boiled yeast-juice was much more 
effective, quantities of 20 to 50 c.c. of yeast-juice mixed with an equal 
volume of boiled juice, which in some experiments was concentrated, 
yielding with O'4, I, and 2 grams of dihydroxyacetone almost the 
theoretical amount of carbon dioxide and alcohol in excess of that 
evolved in the absence of this substance. It was further observed that 
the fermentation of this substance commenced much more slowly than 
that of glucose. No explanation of either of these facts has at present 
been offered. The conclusion drawn from their experiments by 
Buchner and Meisenheimer that dihydroxyacetone is readily ferment- 
able, was confirmed by Lebedeff [1911, i], who further made the im- 
portant observation that during the fermentation of dihydroxyacetone 
the same hexosephosphoric acid is produced as is formed during the 
fermentation of the hexoses. Lebedeff accordingly propounded 
a scheme of alcoholic fermentation according to which the hexose 



106 ALCOHOLIC FERMENTATION 

was first converted into two molecules of triose, the latter being 
first esterified to triosephosphoric acid and then condensed to hex- 
osediphosphoric acid, which then underwent fermentation, after being 
hydrolysed to phosphoric acid, and some unidentified substance, pro- 
bably an unstable modification of a hexose, much more readily 
attacked by an appropriate enzyme than the original glucose or fruct- 
ose [1911, i, pp. 2941-2]. 

The idea that the sugar is first converted into triose and this into 
triosemonophosphoric acid had been previously suggested by IwanofT 
who postulated the agency of a special enzyme termed synthease [ 1 909, 
i], and supposed that this triosemonophosphoric acid was then directly 
fermented to alcohol, carbon dioxide and phosphoric acid. According 
both to Iwanoff and Lebedeff the phosphoric ester is an intermediate 
product and its decomposition provides this sole source of carbon 
dioxide and alcohol. This is quite inconsistent with the facts re- 
counted above (Chap. Ill), which prove that the formation of the 
hexosephosphate is accompanied by an amount of alcoholic fermentation 
exactly equivalent to the quantity of hexosephosphate produced, and 
that the rate of fermentation rapidly falls as soon as the free phosphate 
has disappeared, in spite of the fact that at that moment the concen- 
tration of the hexosephosphate is at its highest, whereas according 
to IwanofFs theory it is precisely under these conditions that the 
maximum rate of fermentation should be maintained. 

It has also been shown that the arguments adduced by Iwanoff in 
favour of the existence of his synthease are not valid [Harden and 
Young, 1910, i]. 

The fermentation of dihydroxyacetone was moreover proved by 
Harden and Young [1912] to be effected by yeast-juice and macera- 
tion extract at a much slower rate than that of the sugars, in spite of 
the fact that the addition of dihydroxyacetone did not inhibit the 
sugar fermentation. The same thing has been shown for living yeast 
by Slator [1912] in agreement with the earlier results of Buchner 
[1910] and Buchner and Meisenheimer [1910]. 

The logical conclusion from LebedefFs experiments would ap- 
pear rather to be that dihydroxyacetone is slowly condensed to a 
hexose and that this is then fermented in the normal manner [Harden 
and Young, 1912; Buchner and Meisenheimer, 1912; Kostytscheff, 
1912, 2]. Buchner and Meisenheimer, however, regard this as improb- 
able on the ground that dihydroxyacetone, being symmetric in con- 
stitution, would yield an inactive hexose of which only at most 50 per 
cent, would be fermentable. Against this it may be urged, however, 



CHEMICAL CHANGES INVOLVED IN FERMENTATION 107 



that enzymic condensation of dihydroxyacetone might very probably 
occur asymmetrically yielding an active and completely fermentable 
hexose. Buchner and Meisenheimer, however, still support the view 
that dihydroxyacetone forms an intermediate stage in the fermentation 
of glucose and adduce as confirmatory evidence of the probability of 
such a change the observation of Fernbach [1910] that this compound 
is produced from glucose by a bacillus, Tyrothrix tenuis, which effects 
the change both when living and after treatment with acetone. 

The balance of evidence, however, appears to be in favour of the 
opinion that dihydroxyacetone does not fulfil the conditions laid 
down by Slator (see p. 103) as essential for an intermediate product 
in the process of fermentation [see also Lob, 1910]. 

Lebedeff subsequently [1912, 4; Lebedeff and Griaznoff, 1912] 
extended his experiments to glyceraldehyde and modified his theory 
very considerably. Using maceration extract it was found in general 
agreement with the results of Buchner and Meisenheimer (p. 105) that 
20 c.c. of juice were capable of producing about half the theoretical 
amount of carbon dioxide from 0*2 gram of glyceraldehyde, whereas 
0*4 gram caused coagulation of the extract and a diminished 
evolution of carbon dioxide. The addition of phosphate diminished 
rather than increased the fermentation. Even in the most favourable 
concentration however (0*2 gram per 20 c.c.) the glyceraldehyde is 
fermented much more slowly than dihydroxyacetone or saccharose, 
as is shown by the following figures : 



20 c.c. Extract 
+ o'2gram. 


CO a in grams in successive 
periods of 


Duration of 
fermentation. 


Total C0 2 . 


Cane sugar 
Dihydroxyacetone 
Glyceraldehyde 


6 hours. 


1 8 hours. 


24 hours. 


6 
6 

48 


0-05 
0-042 
0-035 


0-050 
0*042 
0-008 


o-ooo 

O'OOO 
0-022 


O'OOO 
O'OOO 

0-005 



Further, during an experiment in which 0-129 gram of CO 2 was evolved 
in 22-5 hours from 0*9 gram of glyceraldehyde in presence of phosphate, 
no change in free phosphate was observed, whereas in a similar experi- 
ment with glucose a loss of about 0*2 gram of P 2 O 5 would have occurred. 
Hence the fermentation takes place without formation of hexosedi- 
phosphate. This was confirmed by the fact that the osazone of hex- 
osephosphoric acid was readily isolated from the products of fermenta- 
tion of dihydroxyacetone (0-259 gram of CO 2 having been evolved in 
twenty hours) but could not be obtained from those of glyceraldehyde 
(0-138 gram CO 2 in twenty hours). 



tog ALCOHOLIC FERMENTATION 

This result is extremely interesting, although it is not impossible 
that the rate of fermentation of the glyceraldehyde is so slow that any 
phosphoric ester produced is hydrolysed as rapidly as it is formed. 

Lebedefif regards the experiments as proof that phosphate takes no 
part in the fermentation of glyceraldehyde and bases on this conclusion 
and his other work the following theory of alcoholic fermentation. 

1. The sugar is split up into equimolecular proportions of glycer- 
aldehyde and dihydroxyacetone : 

(a) C 6 H 12 6 = C 3 H 6 3 + C,H 6 8 . 

2. The dihydroxyacetone then passes through the stages previously 
postulated (p. 106). 

(b) 4 C 3 H 6 3 + 4 R 2 HP0 4 = 4 C 3 H 5 O 2 PO 4 R, + 4 H 2 O. 

(c) 4 C 3 H 6 2 P0 4 R 2 = 2 C 6 H 10 4 (R 2 P0 4 ) 2 . 

(d) 2 C 6 H 10 4 (R 2 P0 4 ) 2 + 4 H 2 = 2 C 6 H 12 6 + 4 R 2 HPO 4 . 

After which the hexose, C 6 H 12 O 6 re-enters the cycle at (a). 

3. The fermentation of the glyceraldehyde occurs according to 
the scheme developed by Kostytschefif (p. 109), pyruvic acid being 
formed along with hydrogen and then decomposed into carbon dioxide 
and acetaldehyde, which is reduced by the hydrogen. Lebedefif, 
however, suggests [1914, I, 2] that glyceric acid is first formed (i) and 
then converted by an enzyme, which he terms dehydratase into pyruvic 
acid (2) : 

(1) CH 2 (OH) . CH(OH) . CHO_CH 2 (OH) . CH(OH) . CH(OH) 2 -CH 2 (OH) . CH(OH) . COOH 

+ H 2 O. + 2H 

(2) CH 2 (OH) . CH(OH) . COOH = CH 3 . CO . COOH + H 2 O. 

The experimental basis for this idea is the fact that glyceric acid is 
fermented by dried yeast and maceration juice [compare Neuberg and 
Tir, 1911]. 

This scheme has the merit of recognising the fact that the carbon 
dioxide does not wholly arise from the products of decomposition of 
hexosephosphate, nor from its direct fermentation. The function 
assigned to the phosphate is that of removing dihydroxyacetone 
and thus preventing it from inhibiting further conversion of hexose 
into triose, according to the reversible reaction 

C 6 H 12 6 =^ 2 C 3 H 6 3 . 

This however appears to be quite inadequate, since, on the one hand, 
the fermentation of glucose proceeds quite freely in presence of as much 
as 5 grams per 100 c.c. of dihydroxyacetone [Harden and Young, 1912], 
and on the other hand alcoholic fermentation appears not to proceed 
at all in the absence of phosphate (see p. 55). This forms the chief 
objection to the theory in its present form. The slow rate at which 



CHEMICAL CHANGES INVOLVED IN FERMENTATION 109 

glyceraldehyde is fermented also affords an argument against the 
validity of Lebedeff's view, but this may possibly be accounted for to 
some extent by the fact that glyceraldehyde is a strong inhibiting agent 
so that it might be more rapidly fermented if added in a more dilute 
condition. 

The unfermented glyceraldehyde cannot be recovered from the 
solution and nothing is known as to its fate except that it readily gives 
rise both to lactic acid and glycerol [Oppenheimer, 1914, I, 2], 
Evidently the reaction between glyceraldehyde and yeast-juice is by 
no means a simple one. 

The Pyruvic Acid Theory. 

The third stage of Lebedeff's theory postulates the intermediate 
formation of pyruvic acid. This idea immediately suggested itself 
when it became known that yeast was capable of rapidly decomposing 
tf-ketonic acids with evolution of carbon dioxide [see Neubauer and 
Fromherz, 1911, p. 350; Neuberg and Kerb, 1912,4; Kostytscheff, 
1912, 2]. 

This scheme has been differently elaborated by different 
workers. According to Kostytscheff it involves (i) the production of 
pyruvic acid from the hexoses, a process accompanied by loss of hy- 
drogen ; (2) the decomposition of pyruvic acid into acetaldehyde and 
carbon dioxide ; and (3) the reduction of the acetaldehyde to ethyl 
alcohol. 

(1) C 6 H 12 6 = 2 CH 3 . CO . COOH + 4[H]. 

(2) 2 CH 3 . CO . COOH = 2 CH 3 . CHO + 2 CO a . 

(3) 2 CH 3 . CHO + 4 H = 2 CH 3 . CH 2 . OH. 

i. As regards the production of pyruvic acid from the hexoses by 
yeast, the only direct evidence is afforded by the experiments of 
Fernbach and Schoen [1913] who have obtained a calcium salt having 
the qualitative properties of a pyruvate by carrying out alcoholic 
fermentation by yeast in presence of calcium carbonate, but have not 
yet definitely settled either the identity of the acid or its origin from 
sugar. Pyruvic acid is, however, very closely related to several sub- 
stances which are intimately connected both chemically and biochemi- 
cally with the hexoses. Thus lactic acid is its reduction product, 

CH 3 .CO.COOH _> CH 3 .CH(OH).COOH, 

+ 2H 

glyceraldehyde can readily be converted into it by oxidation to glyceric 
acid followed by abstraction of water (Erlenmeyer), 



no ALCOHOLIC FERMENTATION 

CH 2 (OH).CH(OH).CHO -> CH 2 (OH) . CH(OH) . COOH -> CH 3 .CO.COOH, 
+ - H 2 ' 

and finally methylglyoxal CH 3 . CO . CHO is its aldehyde. 

2. The decomposition of pyruvic acid into acetaldehyde and car- 
bon dioxide has already been fully discussed (Chapter VI). The uni- 
versality of the enzyme carboxylase in yeasts and the rapidity of its 
action on pyruvic acid form the strongest evidence at present available 
in favour of the pyruvic acid theory. Given the pyruvic acid, there is 
no doubt that yeast is provided with a mechanism capable of decom- 
posing it at the same rate as an equivalent amount of sugar. 

3. The final step postulated by the pyruvic acid theory is the 
quantitative reduction to ethyl alcohol of the acetaldehyde formed 
from the pyruvic acid. 

The idea that acetaldehyde is an intermediate product in the 
various fermentations of sugar has frequently been entertained 
[Magnus Levy, 1902 ; Leathes, 1906 ; Buchner and Meisenheimer, 
1908 ; Harden and Norris, D., 1912] although no very definite experi- 
mental foundation exists for the belief. It is, however, a well-known 
fact that traces of acetaldehyde are invariably formed during alcoholic 
fermentation [see Ashdown and Hewitt, 1910], and this is of course 
consistent with the occurrence of acetaldehyde as an intermediate pro- 
duct. Important evidence as to the specific capability of yeast to re- 
duce acetaldehyde to alcohol has been obtained by several workers. 
Thus Kostytscheff[i9i2, 3 ; Kostytscheff and Hubbenet, 1913] found 
that pressed yeast, dried yeast and zymin all reduced acetaldehyde to 
alcohol, 50 grams of yeast in 10 hours producing from 660 mg. of alde- 
hyde 265 mg. of alcohol in excess of the amount produced by auto- 
fermentation in absence of added aldehyde. Maceration extract was 
found to reduce both in absence and in presence of sugar, whereas 
Lebedeff and Griaznoff [1912] obtained no reduction in presence of 
sugar, and observed that the power of reduction was lost by the extract 
on digestion, a circumstance which suggests the co-operation of a co- 
enzyme in the process. Neuberg and Kerb [1912, 4 ; 1913, i] have 
also been able to show by large scale experiments that alcohol is pro- 
duced in considerable quantity by the fermentation of pyruvic acid by 
living yeast in absence of sugar and that the yield is increased by the 
presence of glycerol. When treated with 22 kilos, of yeast, I kilo, of 
pyruvic acid yielded 241 grams of alcohol in excess of that given by 
the yeast alone, whilst in presence of glycerol the amount was 360 
grams, the amount theoretically obtainable being 523 grams. The func- 
tion of the glycerol is not understood but is probably that of lessening 
the rate of destruction of the yeast enzymes. 



CHEMICAL CHANGES INVOLVED IN FERMENTATION 1 1 1 

That yeast possesses powerful reducing properties has long been 
known and many investigations have been made as to the relation of 
these properties to the process of alcoholic fermentation. Thus Hahn 
(Buchner, E. and H., and Hahn, 1903, p. 343) found that the power 
of reducing methylene blue was possessed both by yeast and zymin and 
on the whole ran parallel to the fermenting power in the process of 
alcoholic fermentation. The intervention of a reducing enzyme was sug- 
gested by Griiss [1904, 1908, 1, 2] and was supported by Palladin [1908]. 
The latter observed that zymin which reduces sodium selenite and 
methylene blue in absence of sugar almost ceases to do so in presence of a 
fermentable sugar, and concluded that the great diminution of reduction 
during fermentation was due to the fact that the reducing enzyme was 
largely combined with a different substrate arising from the sugar, the 
reduction of which was necessary for alcoholic fermentation. Griiss, 
however, found that with living yeast the reduction is greatly increased 
in presence of a fermentable sugar, while Harden and Norris, R. V. 
[1914] confirmed the observation of Griiss, but found that the reducing 
power of zymin is not seriously affected by the presence of a ferment- 
able sugar in concentration less then 20 grams per 100 c.c., whilst its fer- 
menting power for glucose is inhibited by I per cent, sodium selenite. 
Hence Palladin's conclusion cannot be regarded as proved. 

Interesting attempts have been made by Kostytscheff and later by 
Lvoff to obtain evidence of the participation of a reductase in alcoholic 
fermentation by adding some substance which would be capable 
either of taking up hydrogen and thus preventing the reduction of the 
acetaldehyde or of converting the aldehyde into some compound less 
liable to reduction. 

Kostytscheff [1912,1; 1913,1,2; 1914; Kostytscheff and Hiibbenet, 
1913; Kosty tscheff and Scheloumoff, 1913 ; Kostytscheff and Brilliant, 
1913] has examined the effect of the addition of zinc chloride, chosen 
with the idea that it might polymerise the aldehyde and thus remove 
it from the sphere of action. As pointed out by Neuberg and Kerb 
[1912, i] this action is not very probable, and it was subsequently 
found [Kostytscheff and Scheloumoff, 1913] that the effect of added 
zinc salts was more probably specifically due to the zinc ion. Fer- 
mentation of sugar by dried yeast still proceeds when O'6 gram of ZnCl 2 
is added to 10 grams of the yeast and 50 c.c. of water, whereas it ceases 
in the presence of I '2 gram of ZnCl 2 . Even the addition of 0*075 gram 
however greatly diminishes the rate of fermentation and the total 
amount of sugar decomposed. The most noteworthy effect is that the 
production of acetaldehyde is increased both in autofermentation and 



H2 ALCOHOLIC FERMENTATION 

in sugar fermentation. The course of the reaction is further modified 
in the sense that the percentage of sugar used up which can be ac- 
counted for in the products decreases, in other words the " disappearing 
sugar" (p. 31) increases. In long continued fermentations more- 
over and particularly with high concentrations of zinc chloride less 
alcohol is produced than is equivalent to the carbon dioxide evolved. 
The interpretation of these results is difficult. Kostytscheff takes 
them to mean (i) that the zinc salt modifies one stage of the reaction 
so that a higher concentration of intermediate products is obtained, and 
(2) that the carbon dioxide and alcohol must be produced at different 
stages or their ratio, in the absence of secondary changes, would be 
unalterable. 

Alternative interpretations are, however, by no means excluded. 
Thus Neuberg and Kerb [1912, I ; 1913, 2] do not regard it as conclus- 
ively proved that the aldehyde really arises from the sugar since they 
have observed its production in maceration extract free from autofer- 
mentation. The method used by Kostytscheff for the separation of 
alcohol and aldehyde (treatment with bisulphite) has also proved un- 
satisfactory in their hands and the results obtained as to the reduction of 
acetaldehyde by yeast, etc., are not accepted. They also consider that 
in any case the small amounts produced (less than 0*2 per cent of the 
sugar used) would not afford convincing evidence that the aldehyde is 
an intermediate product, although it must be admitted that no large ac- 
cumulation of an intermediate product could be reasonably expected. 
It may also be pointed out that the increase in "disappearing sugar" 
may be simply due to the fact that in the controls the whole of the 
sugar was fermented, so that any polysaccharide formed at an earlier 
stage would have been hydrolysed and fermented, whereas in the 
presence 'of zinc chloride excess of sugar was present throughout 
the whole experiment. 

Lvoff [1913, I, 2, 3] has made quantitative experiments on the effect 
of methylene blue both on the sugar fermentation and autofermentation 
of dried yeast and maceration extract. In presence of sugar the 
methylene blue causes a decrease in the extent of fermentation, the 
difference during the time required for reduction of the methylene blue 
being represented by an amount of glucose equimolecular to the latter. 
In the absence of sugar on the other hand an excess of carbon dioxide 
equimolecular to the methylene blue is evolved but no corresponding 
increase in the alcohol production occurs. The effect of methylene 
blue is evidently complex and it is impossible at present to say 
whether Lvoff's contention is correct that the methylene blue actually 



CHEMICAL CHANGES INVOLVED IN FERMENTATION 113 

interferes with the fermentation by taking up hydrogen (2 atoms per 
molecule of glucose) destined for the subsequent reduction of some 
intermediate product or whether the effect is one of general depres- 
sion of the fermenting power which would be presumably proportional 
to the concentration of methylene blue and inversely proportional to 
that of the fermenting complex [see Harden and Norris, R. V., 1914]. 
In any case it will be noticed that Lvoffs interpretation of the results 
is at variance with the requirements of Kostytscherf s theory (p. 109) 
according to which 4 atoms of hydrogen should be given off by a 
molecule of glucose. 

Kostytscheff [1913, 2; Kostytscheff and Scheloumoff, 1913] has 
also observed a depression of the extent of fermentation by methylene 
blue without any serious alteration in the ratio of CO 2 to alcohol, 
although an increase occurs in the production of acetaldehyde. 

On the whole it cannot be said that the evidence gathered from 
experiments on the reduction of acetaldehyde and methylene blue is 
very convincing. All that is established beyond doubt seems to be 
that yeast possesses a reducing mechanism for many aldehydes [see 
also in this connection Lintner and Luers, 1913; Lintner and von 
Liebig, 1911; as well as Neuberg and Steenbock, 1913, 1914] and 
colouring matters. This mechanism appears to be capable of activity 
in the absence of sugar and it is to be supposed that in accordance with 
the views of Bach [191 3] the necessary hydrogen is derived from water 
and that some acceptor for the oxygen simultaneously liberated is also 
present. There seems however at the moment to be no sufficient reason 
to suppose that this mode of reduction is in any way altered by the 
presence of sugar and until the production of intermediate products 
equivalent to the amount of substance reduced is actually demonstrated, 
the conclusions of these workers may be regarded as not fully justified. 

Neuberg and Kerb [1913, 2] themselves tentatively propose a 
complicated scheme possessing some novel features according to which 
methylglyoxal is the starting-point for the later stages of the change. 

(a) A small portion of this is converted by a reaction which may 
be variously interpreted as a Cannizzaro transformation or a reductase 
reaction into glycerol and pyruvic acid. 

CH 2 : C(OH) . CHO + H 2 O K, CH 2 (OH) . CH(OH) . CH 2 (OH) 

glycerol 



i 



CH 2 :C(OH).CHO O CH 2 : C(OH) . COOH 

Pyruvic acid 

(&) The pyruvic acid is then decomposed by carboxylase yielding 
aldehyde and carbon dioxide (equation 2, p. 109). 

8 



H4 ALCOHOLIC FERMENTATION 

(c) The aldehyde and a molecule of glyoxal then undergo a Can- 
nizzaro reaction and yield alcohol and pyruvic acid, 

CHg.CO.CHO O CH 3 .CO.COOH 

+ | = + 

CH 3 . CHO H 2 CH 3 . CH 2 (OH) 

and the latter then undergoes reaction (b). 

A small amount of glycerol is thus necessarily formed, as is actu- 
ally found to be the case. 

The experimental foundation for stages (a) and (c) will be awaited 
with great interest, as well as the proof that methylglyoxal is readily 
fermentable (see p. 104). 

The Formic Acid Theory. 

An interesting interpretation of the phenomena of fermenta- 
tion was attempted by Schade [1906] based upon the conception that 
glucose under the influence of catalytic agents readily decomposes into 
acetaldehyde and formic acid. It was subsequently found that the ex- 
perimental evidence upon which this conclusion was founded had been 
wrongly interpreted [Buchner, Meisenheimer, and Schade, 1906 ; Schade, 
1907], but Schade has succeeded in devising an interesting series of 
reactions by means of which alcohol and carbon dioxide can be obtained 
from sugar by the successive action of various catalysts. The following 
are the stages of this series: (i) Glucose, fructose, and mannose are 
converted by alkalis into lactic acid along with other products. (2) 
Lactic acid when heated with dilute sulphuric acid yields a mixture of 
acetaldehyde and formic acid : 

CH 3 . CH(OH) . COOH = CH 3 . CHO + H . COOH. 

(3) It has long been known that formic acid is catalysed by metallic 
rhodium at the ordinary temperature into hydrogen and carbon dioxide, 
and Schade has found that when a mixture of acetaldehyde and formic 
acid is submitted to the action of rhodium the acetaldehyde is reduced 
to alcohol at the expense of the hydrogen and the carbon dioxide 
is evolved : 

CH 3 . CHO + H . COOH = CH 3 . CH 2 (OH) + CO 2 . 

Schade suggests [1908] that the fermentation of sugar may proceed by 
a similar series of reactions catalysed by enzymes, the acetaldehyde 
and formic acid being derived not from the relatively stable lactic acid 
but more probably from a labile substance capable of undergoing 
change either into lactic acid or into aldehyde and formic acid. 

It will be noticed that this theory resembles the pyruvic acid 



CHEMICAL CHANGES INVOLVED IN FERMENTATION 115 

theory in postulating the immediate formation of acetaldehyde but 
differs from it by supposing that the reduction is effected at the expense 
of formic acid produced at the same time. 

The acetaldehyde question has already been discussed. In view 
of the fact that formic acid is a regular product of the action of many 
bacteria on glucose [see Harden, 1901], Schade's theory of alcoholic 
fermentation may be said to be a possible interpretation of the facts. 
Formic acid is known to be present in small amounts in fermented 
sugar solutions and the actual behaviour of yeast towards this 
substance has been investigated in some detail by Franzen and 
Steppuhn [1911 ; 1912, I, 2], who have obtained results strongly re- 
miniscent of those obtained with lactic acid by Buchner and Meisen- 
heimer (p. 102). Many yeasts when grown in presence of sodium 
formate decompose a certain proportion of it, whereas in absence of 
formate they actually produce a small amount of formic acid the ab- 
solute quantities being usually of the order of 0-0005 gram molecule 
(O'O23 gram) per 100 c.c. of medium in 4 to 5 days. Only in the case of 
5. validus did the consumption of formic acid in 5 days reach 0*0017 
gram molecule (0*08 gram). Somewhat similar but rather smaller re- 
sults were given by yeast-juice, a small consumption of formic acid 
being usually observed. The possibility thus exists that formic 
acid may be an intermediate product of alcoholic fermentation and 
Franzen argues strongly in favour of this view. 

Direct experiment, on the other hand, shows that yeast-juice can- 
not ferment a mixture of acetaldehyde and formic acid, even when 
these are gradually produced in molecular proportions in the liquid by 
the slow hydrolysis of a compound of the two, ethylideneoxyformate, 
OHC.O. CH(CH 3 ). O.CH(CH 3 ).O.CHO, this method being 
adopted to avoid the inhibiting effect of free acetaldehyde and formic 
acid [Buchner and Meisenheimer, 1910]. Nor is the reduction of acet- 
aldehyde assisted by the presence of formate [Neuberg and Kerb, 
1912, 4; Kostytscheff and Hubbenet, 1912]. 

A modified form of Schade's theory has been suggested by Ash- 
down and Hewitt [1910], who have found that when brewer's 
yeast is cultivated in presence of sodium formate the yield of aldehyde, 
as a rule, becomes less. They regard the aldehyde as derived from 
alanine, CH 3 . CH(NH 2 ). COOH, one of the amino-acids formed from 
the proteins by hydrolysis, which is known to be attacked by yeast 
in the characteristic manner (p. 87), forming alcohol, carbon dioxide, 
and ammonia. Fermentation is supposed to proceed in such a way 
that the sugar is first decomposed into two smaller molecules, C 3 H fi O 3 

8 * 



ii6 ALCOHOLIC FERMENTATION 

(equation i) 3 and that these react with formamide to produce alanine 
and formic acid (ii). The alanine then enters into reaction with formic 
acid, producing alcohol, carbon dioxide, and formamide (iii) : 
(i) C 6 H 12 6 = 2 C 3 H 6 3 . 

(ii) C 3 H 6 O 3 + H . CO . NHj, = CH 3 . CH(NH 2 ) . COOH + H . COOH. 

(iii) CH 3 . CH(NH 2 ) . COOH + H . COOH = CH 3 . CH 2 . OH + CO 2 + H . CO . NH 2 . 

According to this scheme all the sugar fermented passes through the 
form of alanine, and the formic acid acts along with the enzyme as 
catalyst, passing into formamide in reaction (iii) and being regenerated 
in (ii). The alanine is in the first place derived from the hydrolysis of 
proteins, or possibly by the reaction of the C 3 H 6 O 3 group with one of 
the higher amino-acids : 

C 3 H 6 3 +C M H 2M + 1 . CH(NH 2 ) . COOH = C W H W + 1 . CH 2 . OH + CO 2 + CH(NH 2 ) . COOH. 
There is as little positive evidence for this course of events as for 
that postulated by Schade, and the theory suffers from the additional 
disability that the chemical reactions involved have not been realised 
in the laboratory. Direct experiments with yeast-juice, moreover, show 
that a mixture of alanine with formic acid or a formate is not fermented, 
whilst neither the added mixture nor formamide seriously effects the 
action of the juice on glucose. 

Other Theories. 

Among other suggestions may be mentioned that of Kohl [1909] 
who asserts that sodium lactate is readily fermented, whilst Kusseroff 
[1910] holds the view that the glucose is first reduced to sorbitol and 
the latter fermented, in spite of the fact that sorbitol itself in the free 
state is not fermented by yeast. 

The rapid appearance and disappearance of glycogen in the yeast 
cell at various stages of fermentation [see Pavy and Bywaters, 1907 ; 
Wager and Peniston, 1910] has led to the suggestion [Griiss, 1904; 
Kohl, 1907] that this substance is of great importance in fermentation, 
and represents a stage through which all the sugar must pass before 
being fermented. The fact that the formation of glycogen has been ob- 
served in yeast-juice by Cremer [1899], an d that complex carbohydrates 
are also undoubtedly formed (p. 31), are consistent with this theory. 
The low rate of autofermentation of living yeast, which is only a few per 
cent of the rate of sugar fermentation, renders this supposition very im- 
probable (Slator), as does the fact that the fermentation of glycogen by 
yeast-juice is usually slower than that of glucose [see also Euler, 1914]. 

An entirely different explanation of the chemical changes attend- 
ant on alcoholic fermentation has been suggested by Lob [1906; 



CHEMICAL CHANGES INVOLVED IN FERMENTATION 117 

1908, 1,2; 1909, 1,2,3,4; i9io;L6bandPulvermacher, 1909], founded 
on the idea that the various decompositions of the sugar molecule both 
by chemical and biological agents are to be explained by a reversal of 
the synthesis of sugar from formaldehyde. As the sugar molecule can 
be built up by the condensation of formaldehyde, so it tends to break 
down again into this substance, and the products observed in any par- 
ticular case are formed either by partial depolymerisation in this sense 
or by partial re-synthesis following on depolymerisation. 

Lob has adduced many striking facts in favour of this view, and 
has shown that very dilute alkalis produce no lactic acid but formal- 
dehyde and a pentose as primary products. These substances represent 
the first stage of depolymerisation and are also formed by the elec- 
trolysis of glucose. 

Lob has himself been unable to detect definite intermediate pro- 
ducts of fermentation by adding reagents, such as aniline, ammonia, 
and phloroglucinol, which would combine with such substances and pre- 
vent their further decomposition [1906]. 

The occurrence of traces of formaldehyde as a product of alcoholic 
fermentation by yeast-juice [Lebedeff, 1908] is at least consistent 
with this theory, but no decisive evidence has so far been obtained 
either for or against it. 

In all the foregoing attempts to indicate the probable stages in the 
production of alcohol and carbon dioxide from sugar, a single molecule 
of the sugar forms the starting-point. The facts recounted in Chapter 
III as to the function of phosphates in alcoholic fermentation, which 
are summed up in the equation : 

2C 6 H 12 9 + 2 R 2 HP0 4 - 2C0 2 + 2C 2 H 6 + 2H 2 O + C 6 H 10 O 4 (PO 4 R 2 ) 2 , 

render it in the highest degree probable that two molecules of the sugar 
are concerned. The most reasonable interpretation of this equation 
appears to be that in the presence of phosphate and of the complicated 
machinery of enzyme and co-enzyme two molecules of the hexose, 
or possibly of the enolic form, are each decomposed primarily into 
two groups. 

Of the four groups thus produced, two go to form alcohol and 
carbon dioxide and the other two are synthesised to a new chain of six 
carbon atoms, which forms the carbohydrate residue of the hexose- 
phosphate. The introduction of the phosphoric acid groups may 
possibly occur before the rupture of the original molecules, and may 
even be the determining factor of this rupture, or again this introduc- 
tion may take place during or after the formation of the new carbon 



n8 ALCOHOLIC FERMENTATION 

chain. Sufficient information is not yet available for the exact for- 
mulation of a scheme for this reaction. Such a scheme, it may be 
noted, would not necessarily be inconsistent with the views of Wohl and 
of Buchner as to the way in which the carbon chain of a hexose is broken 
in the process of fermentation, but would interpret differently the sub- 
sequent changes which are undergone by the simpler groups which are 
the result of this rupture. The reaction might thus proceed without 
the formation of definite intermediate products, whilst opportunity 
would be afforded for the production of a small quantity of by-products 
such as formaldehyde, glycerol, lactic acid, acetic acid, etc., by secondary 
reactions. 

A symmetrical scheme can readily be constructed for such a change, 
but much further information is required before any decisive conclusion 
can be drawn as to the precise course of the reaction which actually 
occurs in alcoholic fermentation. 



CHAPTER IX. 

THE MECHANISM OF FERMENTATION. 

THE analysis of the process of alcoholic fermentation by yeast-juice 
and other preparations from yeast which has been carried out in the 
preceding chapters has shown that the phenomenon is one of a very 
complex character. The principal substances directly concerned in the 
change appear to be the enzyme and co-enzyme of the juice, a second 
enzyme, hexosephosphatase, and, in addition, sugar, phosphate, and the 
hexosephosphate formed from these. During autofermentation two 
other factors are involved, the complex carbohydrates of the juice, 
including glycogen and dextrins, and the diastatic ferment by which 
these are converted into fermentable sugars. It is also possible that 
the supply of free phosphate is partially provided by the action of pro- 
teoclastic ferments on phosphoproteins. Under special circumstances 
the rate at which fermentation proceeds may be controlled by the 
available amount of any one of these numerous substances. 

When the juice from well-washed yeast is incubated, the pheno- 
menon of autofermentation is observed. The juice contains an abun- 
dant supply of enzyme, co-enzyme, and phosphate or hexosephosphate, 
and in this case the controlling factor is usually the supply of sugar, 
which is conditioned by the concentration of the diastatic enzyme or of 
the complex carbohydrates as the case may be. When this is the case 
the measured rate of fermentation is the rate at which sugar is being 
produced in the juice, this being the slowest of the various reactions 
which are proceeding under these circumstances. If sugar be now 
added, an entirely different state of affairs is set up. As soon as any 
accumulated phosphate has been converted into hexosephosphate, the 
normal rate of fermentation which is usually higher than that of auto- 
fermentation is attained, and, provided that excess of sugar be present, 
fermentation continues for a considerable period at a slowly diminish- 
ing rate and finally ceases. During the first part of this fermentation 
the rate is controlled entirely by the supply of free phosphate, and this 
depends mainly on the concentration of the hexosephosphatase and of 
the hexosephosphate, and only in a secondary degree on the decom- 

119 



120 ALCOHOLIC FERMENTATION 

position of other phosphorus compounds by other enzymes and on the 
concentration of the sugar. The amount of hexosephosphate in yeast- 
juice is usually such that an increase in its concentration does not 
greatly affect the rate of fermentation, and hence the measured rate 
during this period represents the rate at which hexosephosphate is be- 
ing decomposed, and this in its turn depends on the concentration of 
hexosephosphatase, which is therefore the controlling factor. As 
fermentation proceeds, the concentration of both enzyme and co-enzyme 
steadily diminishes, as already explained, probably owing to the action 
of other enzymes, so that at an advanced stage of the fermentation, 
the controlling factor may be the concentration of either of these, 
or the product of the two concentrations (see p. 122). The hexose- 
phosphatase appears invariably to outlast the enzyme and co-enzyme. 
The condition at any moment could be determined experimentally if 
it were possible to add enzyme, co-enzyme and hexosephosphatase at 
will and so ascertain which of these produced an acceleration of the 
rate. 

Unfortunately this can at present be only very imperfectly accom- 
plished, owing to the impossibility of separating these substances from 
each other and from accompanying matter which interferes with the 
interpretation of the result. 

A third condition can also be established by adding to the ferment- 
ing mixture of the juice and sugar a solution of phosphate. The 
supply of phosphate is now almost independent of the action of the 
hexosephosphatase, and the measured rate represents the rate at which 
reaction (i), p. 51, can occur between sugar and phosphate in the presence 
of the fermenting complex consisting of enzyme and co-enzyme. This 
change is controlled, so long as sugar and phosphate are present in 
the proper amounts, by the concentration of the fermenting complex 
or possibly of either the enzyme or the co-enzyme. If only a single ad- 
dition of a small quantity of phosphate be made, the rate falls as soon 
as the whole of this has been converted into hexosephosphate and the 
reaction then passes into the stage just considered, in which the rate 
is controlled by the production of free phosphate. 

Although these varying reactions have not yet been exhaustively 
studied from the kinetic point of view, owing to the experimental diffi- 
culties to which allusion has already been made, investigations have 
nevertheless been carried out on the effect of the variation of con- 
centration of yeast-juice and zymin as a whole, as well as of the car- 
bohydrate. Herzog [1902, 1904] has made experiments of this kind 
with zymin, and Euler [1905] with yeast-juice, whilst many of the re^ 



THE MECHANISM OF FERMENTATION 121 

suits obtained by Buchner and by Harden and Young are also avail- 
able. 

The actual observations made by these authors show that the in- 
itial velocity of fermentation is almost independent of the concentration 
of sugar within certain limits, but decreases slowly as the concentration 
increases. When the velocity constant is calculated on the assumption 
that the reaction is monomolecular [see Bayliss, 1914, Chap. VI], 
approximate constancy is found for the first period of the fermentation. 
This method of dealing with the results is, however, as pointed out by 
Slator, misleading, the apparent agreement with the law of mono- 
molecular reactions being probably due to the gradual destruction of 
the fermenting complex. 

Experiments with low concentrations of sugar are difficult to in- 
terpret, the influence of the hydrolysis of glycogen and of dextrins on 
the one hand, and the synthesis of sugar to more complex carbo- 
hydrates on the other (p. 3 1 ), having a relatively great effect on the 
concentration of the sugar. Unpublished experiments (Harden and 
Young) indicate, however, that the velocity of fermentation remains 
approximately constant, until a certain very low limit of sugar concen- 
tration is reached, and then falls rapidly. The fall in rate, however, 
only continues over a small interval of concentration, after which the 
velocity again becomes approximately constant and equal to the rate 
of autofermentation. During this last phase, as already indicated, the 
velocity is generally controlled by the rate of production of sugar and 
no longer by that of phosphate, this substance being now present in 
excess. In other words, the rate of fermentation of sugar by yeast- 
juice and zymin is not proportional to the concentration of the sugar 
present as required by the law of mass, but, after a certain low limit of 
sugar concentration, is independent of this and is actually slightly de- 
creased by increase in the concentration of the sugar. 

The relations here are very similar to those shown to exist by 
Duclaux[i899] and Adrian Brown [1902] for the action of invertase on 
cane sugar and are probably to be explained in the manner suggested 
by the latter. According to this investigator, the enzyme unites with 
the fermentable material, or as it is now termed, the substrate or 
zymolyte, forming a compound which only slowly decomposes so that 
it remains in existence for a perceptible interval of time. The 
rate of fermentation depends on the rate of decomposition of this 
compound and hence varies with its concentration. This conception 
leads to the result that the rate of fermentation will increase with the 
concentration of the substrate up to a certain limit and will then remain 



122 ALCOHOLIC FERMENTATION 

constant, unless interfered with by secondary actions. This limit of 
concentration is that at which there is just sufficient of the material in 
question present to combine with practically the whole of the enzyme, 
so that no further increase in its amount can cause a corresponding 
increase in the quantity of its compound with the enzyme or in the 
rate of fermentation which depends on the concentration of that com- 
pound. 

The curve relating the rate of action of such an enzyme with the 
concentration of the zymolyte therefore consists of two portions, one 
in which the rate at any moment is proportional to the concentration 
of the zymolyte, according to the well-known law of the action of mass, 
and a second in which the rate at any moment is almost independent 
of that concentration, approximately equal amounts being decomposed 
in equal times whatever the concentration of the substrate. 

The results of the experiments with yeast-juice therefore indicate 
that what is being measured is a typical enzyme action, but afford no 
information as to which of the many possible actions is the controlling 
one, a fact which must be ascertained for each particular case in the 
manner indicated above. 

Clowes [1909], using washed zymin free from fermenting power 
and adding various volumes of boiled yeast extract, found that the 
velocity of reaction was proportional to the product of the concen- 
trations of zymin and yeast extract up to a certain optimum con- 
centration. He interprets these concentrations as representing the 
concentrations of zymase and co-enzyme, but they also represent 
the concentrations of hexosephosphatase (present in the zymin) and 
phosphate (present in the yeast extract), so that at least four factors 
were being altered instead of only two. 

It has already been mentioned that Euler and Kullberg [1911, 3] 
found the conversion of phosphate into hexosephosphate in presence of 
excess of glucose to proceed according to a monomolecular reaction 

(P. 5 8). 

The rate of fermentation is diminished by dilution of the yeast- 
juice, but less rapidly than the concentration of the juice. Herzog 
found that when the relation between concentration of enzyme and 
the velocity constant of the reaction is expressed by the formula 
K 1 /K 2 = (C 1 /C 2 ) M where Kj and K 2 are the velocity constants corres- 
ponding with the enzyme concentrations Q and C 2 , the value for n is 
2 for zymin, whilst Euler working with yeast-juice obtained values 
varying from I '29 to I -67 and decreasing as K increased. 

The temperature coefficient of fermentation by zymin was found 



THE MECHANISM OF FERMENTATION 123 

by Herzog to be K 24 . 5 o/K 14 . 5 = 2'88, which agrees well with the 
value found by Slator for yeast-cells (p. 1 29). 

When we endeavour to apply the results of the investigations of 
the fermentation of sugar by yeast-juice, zymin, etc., to the process 
which goes on in the living cell, considerable difficulties present them- 
selves. A scheme of fermentation in the living cell can, however, 
easily be imagined, which is in harmony with these results. According 
to the most simple form of this ideal scheme, the sugar which has 
diffused into the cell unites with the fermenting complex and under- 
goes the characteristic reaction with phosphate, already present in the 
cell, yielding carbon dioxide, alcohol, and hexosephosphate. The 
latter is then decomposed, just as it is in yeast-juice, but more rapidly, 
and the liberated phosphate again enters into reaction, partly with the 
sugar formed from the hexosephosphate and partly with fresh sugar 
supplied from outside the cell. The main difference between ferment- 
ation by yeast-juice and by the living cell would then consist in the 
rate of decomposition of the hexosephosphate, for it has been shown 
that yeast-juice in presence of sufficient phosphate can ferment sugar 
at a rate of the same order of magnitude (from 30 to 50 per cent.) as that 
attained by living yeast. 

The difference between the two therefore would appear to lie not 
so much in their content of fermenting complex as in their very different 
capacity for liberating phosphate from hexosephosphate and thus 
supplying the necessary conditions for fermentation. 

A simple calculation based on the phosphorus content of living 
yeast [Buchner and Haehn, 1910, 2] shows that the whole of this 
phosphate must pass through the stage of hexosephosphate every five or 
six minutes in order to maintain the normal rate of fermentation, 
whereas in an average sample of yeast-juice the cycle, calculated in 
the same way, would last nearly two hours. 

Wherein this difference resides is a difficult question, which cannot 
at present be answered with certainty. 

In the first place it must be remembered that a very great acceler- 
ation of the action of the hexosephosphatase is produced by arsenates 
(p. 79), and this suggests the possibility that some substance possess- 
ing a similar accelerating power is present in the yeast-cell and is lost 
or destroyed in the various processes involved in rendering the yeast 
susceptible to phosphate. The great variety of these processes- 
extraction of yeast-juice by grinding and pressing, drying and macer- 
ating, heating, treating with acetone and with toluene renders this 
somewhat improbable, and so far no such substance has been detected. 



I2 4 ALCOHOLIC FERMENTATION 

A comparison of living yeast, zymin, and yeast-juice shows that 
these are situated on an ascending scale with respect to their response 
to phosphate. Taking fructose as the substrate in each case, yeast 
does not respond to phosphate at all (Slator), the rate of fermentation 
by zymin is approximately doubled (p. 46), and that by yeast-juice 
increased ten to forty times, whilst the maximum rates are in each case 
of the same order of magnitude. Euler and Kullberg, however, have 
observed an acceleration of about 25 per cent in the rate of fermenta- 
tion of yeast in presence of a 2 per cent, solution of monosodium phos- 
phate, NaH 2 PO 4 [i9H, 1,2]. 

The high rate of fermentation by living yeast and its lack of 
response to phosphate may possibly be explained by supposing that 
the balance of enzymes in the living cell is such that the supply of 
phosphate is maintained at the optimum, and the rate of fermentation 
cannot therefore be increased by a further supply. 

A further difference lies in the fact that yeast-juice and zymin 
respond to phosphate more strongly in presence of fructose than of 
glucose, whereas yeast ferments both sugars at the same rate (p. 131 ), 
and this property has been shown to be connected with the specific 
relations of fructose to the fermenting complex. It seems possible 
that these differences are associated with the gradual passage from the 
complete living cell of yeast, through the dead and partially dis- 
organised cell of zymin to yeast-juice in which the last trace of cellular 
organisation has disappeared and the contents of the cell are uniformly 
diffused throughout the liquid. Living yeast is, moreover, not only 
unaffected by phosphate but only decomposes hexosephosphate ex- 
tremely slowly (Iwanoff). 

Some light is thrown on these interesting problems by the effect 
of antiseptics on fermentation by yeast-cells and by yeast-juice. The 
action of toluene has hitherto been most completely studied, and this 
substance is an extremely suitable one for the purpose since it has 
practically no action whatever on fermentation by yeast-juice. The 
experiments of Buchner have, in fact, shown that the normal rate of 
fermentation and the total fermentation produced, are almost unaffected 
by the presence of toluene even in the proportion of I c.c. to 20 c.c. 
of yeast-juice. What then is the effect of toluene on the living yeast- 
cell? When toluene in large excess is agitated with a fermenting 
mixture of yeast and sugar, the rate of fermentation falls rapidly at 
first and then more slowly until a relatively constant rate is attained 
which gradually decreases in a similar manner to the rate of fermenta- 
tion by yeast-juice. Thus at air temperature (16) 10 grams of 



THE MECHANISM OF FERMENTATION 



125 



yeast suspended in 50 c.c. of 6 per cent, glucose solution gave the 
following results when agitated with toluene : 



Time after Addition of Toluene 
Minutes. 


C.c. of CO 2 per Minute. 


Time. 


C.c. per 

Minute. 


o 


4-6 


6 


1-6 


I 


4 


8 


I'2 


2 


3'3 


12 


0-85 


3 


2-6 


24 


0-8 


4 


2 


32 


0-5 


5 


1-8 




constant 



Simultaneously 'with this, the yeast acquires the property of decom- 
posing and fermenting hexosephosphate and of responding to the 
addition of phosphate. This last property is only acquired to a small 
degree in this way but it becomes much more strongly developed if the 
pressed yeast be washed with toluene on the filter pump. Thus 10 
grams of yeast after this treatment fermented fructose at 1-2 c.c. per 
three minutes; after the addition of phosphate (5 c.c. of O'6 molar 
phosphate) the rate rose to 6-9 and then gradually fell in the typical 
manner [Harden, 1910; see also Euler and Johansson, 1912, 3]. 

The current explanation of the great decrease in rate of ferment- 
ation which attends the action of toluene and other antiseptics on living 
yeast, and also follows upon the disintegration of the cell, appears to 
be that in living yeast the high rate of fermentation is maintained by 
the continued production of relatively large fresh supplies of fermenting 
complex, and that when the power of producing this catalytic agent is 
destroyed by the poison, the rate of fermentation falls to a low value, 
corresponding to the store of zymase still present in the cell (cf. 
Buchner, E. and H., and Hahn, 1903, pp. 176, 180). 

This explanation implies that the rate of fermentation after the 
action of the toluene represents the amount of fermenting complex 
present, a supposition which has been shown (p. 53) to be highly im- 
probable. It further necessitates, as also pointed out independently by 
Euler and Ugglas [1911], a rapid destruction of the fermenting complex 
both in the process of fermentation and by the action of the antiseptic, 
as otherwise the store of zymase remaining in the dead cell would be 
practically the same as that contained in the living cell at the moment 
when it was subjected to the antiseptic, and this store would therefore 
suffice to carry out fermentation at the same rate in the dead 
as in the living cell. No such rapid destruction, however, occurs 
in yeast-juice, as judged by the rate of fermentation, which falls off 



126 ALCOHOLIC FERMENTATION 

slowly and to about the same extent in the presence or absence 
of toluene. Moreover, as shown above, it is highly probable that the 
actual amount of fermenting complex in yeast-juice is a large fraction 
of that present at any moment in the cell, and is capable under 
suitable conditions of producing fermentation at a rate comparable 
with that of the living cell. 

This last criticism also applies to the view expressed by Euler [Euler 
and Ugglas, 1911 ; Euler and Kullberg, 1911, I, 2] that in the living 
cell the zymase is partly free and partly combined with the protoplasm ; 
when the vital activity of the cell is interfered with, the combined 
portion of the zymase is thrown out of action and only that which was 
free remains active. 

The suggestion made by Rubner [1913] that the action of yeast 
on sugar is in reality chiefly a vital act, but that a small proportion of 
the change is due to enzyme action, is similar in its consequences to 
that of Euler and may be met by the same arguments. Buchner and 
Skraup [1914] have moreover shown that the effects of sodium chloride 
and toluene on the fermenting power of yeast which were observed 
by Rubner, can be explained in other ways. 

Some other explanation must therefore be sought for this phe- 
nomenon. Great significance must be attached in this connection to the 
relation noted above between the degree of disintegration and dis- 
organisation of the cell and the fall in the normal rate of fermentation. 
It seems not impossible that fermentation may be associated in the 
living cell with some special structure, or carried on in some special 
portion of the cell, perhaps the nuclear vacuole described by Janssens 
and Leblanc [1898], Wager [1898, 1911 ; Wager and Penistpn, 1910] 
and others which undergoes remarkable changes both during fermen- 
tation and autofermentation [Harden and Rowland, 1901]. The dis- 
organisation of the cell might lead to many modifications of the 
conditions, among others to the dilution of the various catalytic agents 
by diffusion throughout the whole volume of the cell. As a matter of 
observation the dilution of yeast-juice leads to a considerable diminution 
of the rate of fermentation of sugar, and it is possible that this is one of the 
chief factors concerned. That phenomena of this kind may be involved 
is shown by the remarkable effect of toluene on the autofermentation 
of yeast. Whereas the fermentation of sugar is greatly diminished by 
the action of toluene, the rate of autofermentation, which is carried on 
at the expense of the glycogen of the cell, is greatly increased. In a 
typical case, for example, the autofermentation of I o grams of yeast 
suspended in 20 c.c. of water amounted to 28 c.c. in 4*8 hours 



THE MECHANISM OF FERMENTATION 127 

at 25, whereas the same amount of yeast in presence of 2 c.c. of 
toluene gave 97-6 c.c. in the same time. 

Many salts produce a similar'effect on English top yeasts (in which 
the autofermentation is large) [Harden and Paine, 1912], whereas 
Neuberg and Karczag in Berlin [1911, 2] were unable to observe this 
phenomenon. 

A necessary preliminary of the fermentation of glycogen is its 
conversion by a diastatic enzyme into a fermentable sugar, and it is 
probable that the effect of the disorganisation of the cell by toluene is 
that this enzyme finds more ready access to the glycogen, which is 
stored in the plasma of the cell. No such acceleration of auto- 
fermentation is effected by the addition of toluene to yeast-juice, and 
hence the result is not due to an acceleration of the action of the 
diastatic enzyme on the glycogen. 

This effect of toluene is similar in character to the action of 
anaesthetics on the leaves of many plants containing glucosides and 
enzymes, whereby an immediate decomposition of the glucoside is 
initiated [see H. E. and E. F. Armstrong, 1910]. 

Although as indicated above Euler's theory cannot apply to zymase 
itself, if applied to the hexosephosphatase it would afford a consistent 
explanation of the facts. According to this modified view it would be 
the hexosephosphatase of yeast which existed largely in the combined 
form, so that in extracts, in dried yeast and in presence of toluene only 
the small fraction which was free would remain active. The zymase 
on the other hand would have to be regarded as existing to a large extent 
in the free state so that it would pass into extracts comparatively 
unimpaired in amount and capable under proper conditions (i.e. when 
supplied with sufficient phosphate) of bringing about a very vigorous 
fermentation. The theory of combined and free enzymes is un- 
doubtedly of considerable value, although it cannot be considered as 
fully established. 

Fermentation by Living Yeast. 

Much important information as to the nature of the processes in- 
volved in fermentation has been acquired by the direct experimental 
study of the action of living yeast on different sugars. 

This phenomenon has formed the subject of several investigations 
from the kinetic point of view, and its general features may now be 
regarded as well established. 

The difficulty, which must as far as possible be avoided in 
quantitative experiments of this sort with living yeast, is the alteration 



128 



ALCOHOLIC FERMENTATION 



in the amount or properties of the yeast, due to growth or to 
some change in the cells. This has been obviated in the work of 
Slator [1906] by determining in every case the initial rate of fer- 
mentation, so that the process only continues for a very short 
period, during which any change in the amount or constitution of the 
yeast is negligible. The method has the further advantage that inter- 
ference of the products of the reaction is to a large extent avoided. 
The pressure apparatus already described (p. 29) was employed by 
Slator, the rate of production of carbon dioxide being measured by the 
increase of pressure in the experimental vessel. 

Influence of Concentration of Dextrose on the Rate of 
Fermentation. 

With regard to this important factor it is found that the action of 
living yeast follows the same law as that of most enzymes (p. 121); 
within certain wide limits the rate of fermentation is almost independent 
of the concentration of the sugar. This conclusion has been drawn by 
many previous investigators from their experiments [Dumas, 1874; 
Tammann, 1889; Adrian Brown, 1892; O'Sullivan, 1898, 1899] and 




2345678 
CRAMS OF DEXTROSE PER 100 cc. 

FIG. 8. 



10 



is implicitly contained in the results of Aberson [1903], although he 
himself regarded the reaction as monomolecular. 

Slator, working with a suspension of ten to twelve yeast-cells per 
1/4000 cubic millimetre at 30, obtained the results which are embodied 
in the curve (Fig. 8). 

This shows that, for the amount of yeast in question, the rate of 
fermentation is almost constant for concentrations of glucose between 



THE MECHANISM OF FERMENTATION 129 

I and 10 grams per 100 c.c., but gradually decreases as the concen- 
tration increases. Below I gram per 100 c.c. the rate decreases 
very rapidly with the concentration. 

It follows from this, in the light of what has already been said 
(p. 121), that the action of living yeast on sugar follows the same 
course as a typical enzyme reaction, although in this case, as in that 
of yeast-juice, no information is given as to the exact nature of this 
reaction. 

Influence of the Concentration of Yeast. 

It appears to be well established that, when changes in the quantity 
and constitution of the yeast employed are eliminated, the rate of 
fermentation is exactly proportional to the number of the yeast-cells 
present (Aberson, Slator). This result might be anticipated, as pointed 
out by Slator, from the fact that the fermentation takes place within 
the cell, each cell acting as an independent individual. 

The diffusion of sugar into the yeast-cell which necessarily precedes 
the act of fermentation has been shown by Slator and Sand [1910] to 
occur at such a rate that the supply of sugar is always in excess of the 
amount which can be fermented by the cell. 

Temperature Coefficient of Alcoholic Fermentation 
by Yeast. 

The temperature coefficient of fermentation by living yeast has 
been carefully determined by Slator by measurements of the initial 
rates at a series of temperatures from 5 to 40 C. The coefficient is 
found to be of the same order as that for many chemical reactions, 
but to vary considerably with the temperature, a rise in temperature 
corresponding with a diminution in the coefficient. The following 
values were obtained for glucose; they are independent of the con- 
centration of yeast and glucose, the class of yeast, and presence 
or absence of nutrient salts, and remain the same when inhibiting 
agents are present. Almost precisely the same ratios are obtained 
for fructose and mannose : 

5 2-65 5-6 

10 2'II 3-8 

15 i'8o 2'8 

20 1-57 2-25 

25 i'43 i'95 

30 1-35 1-6 

35 i*2o 

Aberson's result, K* + io/IQ= 272, which represents the mean co- 
efficient for 10 between 12 and 33, agrees well with this. 

9 



130 



ALCOHOLIC FERMENTATION 



Action of Accelerating Agents on Living Yeast. 

Slator [1908, i] was unable to find any agent which greatly 
accelerated the rate of fermentation of living yeast. Small concentra- 
tions of various inhibiting agents which are often supposed to act in 
this way were quite ineffective, and phosphates, which produce such a 
striking change in yeast-juice, were almost without action (cp. p. 124). 

Euler and Backstrom [1912], however, have made the important 
observation that sodium hexosephosphate causes a considerable ac- 
celeration although it is itself neither fermented nor hydrolysed under 
these conditions. The extent of this is evident from the following 
numbers : 



20 c.c. of 20 per cent, glucose solution. 
0-25 g. yeast [Yeast H of St. Erik's brewery]. 


Without addition. 


+ 0*5 g. Na hexosephosphate. 


Time. 
Min. 


C0 2 . 


Time. 
Min. 


co a . 


46 
7 6 
197 
347 
488 


I0'5 
I7'5 
45 
74'5 
95 


37 
73 

188 

321 

450 


8 
19 
52'5 
123 
193*5 



The observation has been confirmed with English top yeast 
(Harden and Young, unpublished experiments), but no explanation of 
the phenomenon is at present forthcoming. 

Euler has also found [Euler and Cassel, 1913 ; Euler and Berggren, 
1912] that yeast extract, sodium nucleinate and ammonium formate 
also increase the rate of fermentation of glucose by yeast, but these 
results have been criticised by Harden and Young [1913] on the 
ground that the possibility of growth of the yeast during the experi- 
ment has not been excluded. 

Fermentation of Different Sugars by Yeast. 

Many valuable ideas as to the nature of fermentation have been 
obtained by a consideration of the phenomena presented by the 
action of yeast on the different hexoses. Of these only glucose, fructose, 
mannose, and galactose are susceptible of alcoholic fermentation by 
yeast, the stereoisomeric hexoses prepared in the laboratory being un- 
fermentable, as are also the pentoses, tetroses, and the alcohols corres- 
ponding to all the sugars. The yeast-cell is therefore much more 
limited in its power of producing fermentation than such an organism 
as, for example, Bacillus coli communis, which attacks substances as 



THE MECHANISM OF FERMENTATION 131 

diverse as arabinose, glucose, glycerol and mannitol, and yields with 
all of them products of the same chemical character, although in varying 
proportions. 

A careful examination of a number of different genera and species 
of the Saccharomycetaceae and allied organisms by E. F. Armstrong 
[1905] has shown that all yeasts which ferment glucose also ferment 
fructose and mannose. Armstrong grew his yeasts in a nutrient solu- 
tion containing the sugar to be investigated, and his experiments are 
open to the criticism that the organisms were hereby afforded an oppor- 
tunity for becoming acclimatised to the sugar. His results, therefore, 
only demonstrate the fact that the organisms in question when culti- 
vated in presence of the sugars examined brought about their ferment- 
ation, and do not exclude the possibility that the same organism when 
grown in presence of a different sugar might not be capable of ferment- 
ing the one to which it had in the other type of experiment become 
acclimatised. 

This has actually been shown to be the case for galactose by Slator 
[1908, i], and it is possible that this circumstance explains the 
negative results obtained by Lindner [1905] with S. exiguus and 
Schizosaccharomyces Pombe upon mannose, a sugar which, according 
to Armstrong, is fermented by both these organisms. 

The same problem has been attacked quantitatively by Slator, 
who has shown that living yeast of various species and genera ferments 
glucose and fructose at approximately the same rate. Moreover, 
when the yeast is acted upon by various inhibiting agents, such as 
heat, iodine, alcohol, or alkalis, the crippled yeast also ferments glucose 
and fructose at the same rate. 

With mannose the relations are somewhat different. The relative 
rate of fermentation of mannose and glucose by yeast is dependent on 
the variety of the yeast and the treatment which it has received. 
Fresh samples of yeast ferment mannose more quickly than glucose, 
but by older samples the glucose is the more rapidly decomposed. 
This is especially the case with yeast, the activity of which has been 
partly destroyed by heat, the relative fermenting power to mannose 
being sometimes reduced by this treatment from 120 per cent, of that 
of glucose to only 12 per cent. (Slator). 

A further difference consists in the fact that with certain yeasts the 
rate of fermentation of glucose is somewhat increased by monosodium 
phosphate whilst that of mannose is unaffected [Euler and Lundeq- 
vist, 1911]. 

Mixtures of glucose and fructose are fermented by yeast at the 

9* 



132 



ALCOHOLIC FERMENTATION 



same rate as either the glucose or the fructose contained in the mix- 
ture would be alone. When, however, mannose and glucose are fer- 
mented simultaneously interference between the reactions takes place, 
and this is especially evident when the yeast has comparatively little 
action on mannose. The following are the results obtained by Slator : 





Relative Rates. 


Yeast. 


2*5 per cent. 


2 '5 per cent. 


2-5 per cent. Glucose + 




Glucose. 


Mannose. 


2 '5 per cent. Mannose. 


S. Thermantitonum 


100 


105 


92 


Brewery yeast, 53 per cent, activity 
destroyed by heat 


IOO 


21 


33 


Brewery yeast, 60 per cent, activity 








destroyed by heat 


zoo 


12 


42 



The case of galactose merits special attention. Previous investi- 
gations [see Lippmann, 1904, p. 734] have shown that the fermentation of 
galactose by yeast differs greatly from that of the other hexoses. The 
subject has been re-investigated by E. F. Armstrong [1905], and 
by Slator [1908, i], Armstrong carried out his experiments in the 
manner already described (p. 131 ), and found that some yeasts had, 
and others had not, the power of fermenting galactose, although all 
were capable of fermenting glucose, fructose, and mannose. 

Slator made quantitative experiments on the same subject. He 
was able to confirm the statement which had previously been made, 
that certain yeasts which have the property of fermenting galactose 
possess it only after the yeast has become acclimatised by culture in 
presence of the sugar. This was shown for brewery yeast and for the 
species mentioned below. This phenomenon is one of great interest 
and is strictly analogous to the adaptation of bacteria which has now 
been quite conclusively established [Neisser, 1906]. 







Relative Rates. 


Yeast 


Mode of Culture 








Glucose. 


Galactose. 


S. Carlsbergensis 



Grown in wort . 
hydrolysed lactose 


IOO 
IOO 


<i 

86, 83, 85, 25, 46, 








51, 69, 54, 155 


S. Cerevisiae . 


wort . 


IOO 


<i 





hydrolysed lactose 


IOO 


21, 26, 2Q 


S. Thermantitonum 


wort . 


IOO 


<I 


M 


hydrolysed lactose 


IOO 


77, 53, 35 


S. Ludwigii 


wort . 


IOO 


<i 


" 


hydrolysed lactose 


IOO 


<i 



THE MECHANISM OF FERMENTATION 



133 



It will be seen that in one case the rate of fermentation of galactose 
was considerably greater than that of glucose. 5. Ludwigii did not 
respond to the cultivation in hydrolysed lactose, but, as Slator points 
out, it is quite possible that repeated cultivation in this medium might 
effect the change, and this would be strictly analogous to the results 
obtained with bacteria. Slator's results have been confirmed by 
Harden and Norris, R. V. [1910], and by Euler and Johansson [1912, 2] 
who have made an exceedingly interesting study of the progress of 
the adaptation. As in the case of mannose the rates of fermentation 
of glucose and galactose are differently affected by agents such as 
heat and alcohol ; moreover, the rate of fermentation of mixtures of 
dextrose and galactose is in no case either the sum or the mean of the 
rates obtained with the separate sugars. The temperature coefficient 
of the fermentation of galactose also differs slightly from that of the 
other hexoses. 



Yeast. 


Relative Rates. 


Glucose. 


Galactose. 


Glucose + Galactose. 




100 
100 
IOO 
100 


o 
34 
155 
76 


103 
103 
119 
124 




S. Carlsbergensis . . . . 
S. Thermantitonum .... 



Assuming that his conclusion that all yeasts which ferment glucose 
also ferment fructose and mannose is correct, Armstrong has drawn 
attention to the fact that these three hexoses are also related by the 
possession of a common enolic form (p. 97) and has suggested that 
this enolic form is the substance actually fermented to carbon dioxide 
and alcohol [1904]. 

The idea that such an intermediate form is the direct subject of 
fermentation has much to recommend it. In the first place it is 
almost certain, as already pointed out, that the sugars in aqueous 
solution do exist, although to a very small extent, in this enolic form. 
The slow rate at which equilibrium is established in aqueous solution, 
however, must be taken as definite evidence that under these circum- 
stances the enolic form is only produced very slowly [compare Lowry, 
1903]. This has been used by Slator [1908, i] as an argument 
against the probability of the preliminary conversion of the sugars into 
the enolic form before fermentation. It appears, however, quite pos- 
sible that under the influence of the fermenting complex of the yeast- 
cell, or of special enzymes, this change might occur much more rapidly, 



134 ALCOHOLIC FERMENTATION 

and at different rates with the different sugars. This reaction might 
in fact control the observed rate of fermentation. This conception 
affords a simple explanation of the different rates of fermentation of 
mannose and glucose, and also of galactose, the enolic form of which 
is quite different, by yeast under different circumstances, but does not 
explain the uniformity of rate observed by Slator for glucose and 
fructose nor the results with mixtures of sugars. The direct ferment- 
ation of a common enolic form is also consistent with the fact that the 
same hexosephosphate is produced from all three hexoses. 

Slator himself prefers the view that the first stage of fermentation 
consists in the rapid combination of the sugar with the enzyme, pro- 
ducing a compound, which then breaks up at a rate which determines 
the observed rate of fermentation. This rate will of course vary with 
the nature of the compound, so that if two sugars form the same com- 
pound they will be fermented at the same rate ; if they form different 
compounds, different rates may result. Slator supposes that glucose 
and fructose form the same compound with the enzyme. This, how- 
ever, appears to involve an intramolecular change of the same order as 
the production of the enolic form, and moreover is not absolutely 
essential, as it is probably sufficient to suppose that the two compounds 
derived from glucose and fructose are very similar, although possibly 
not absolutely identical. Mannose and galactose, on the other hand, 
form stereoisomeric compounds, and the capacity of the fermenting 
complex to form these compounds may be affected by various agents 
to a different extent from its capacity for combining with glucose or 
fructose. 

A third theory has also been suggested to explain these phenomena, 
according to which the various sugars are fermented by different 
enzymes [see Slator, 1908, i]. The uniformity of the result obtained 
with glucose and fructose suggests that these two sugars are fermented 
by the same enzyme (glucozymase), mannose and galactose by different 
ones (mannozymase and galactozymase). This would afford a simple 
explanation of the different rates of fermentation for different sugars 
and of different degrees of sensitiveness towards reagents. 

If, however, a separate and independent mechanism were present 
for each sugar, the rate of fermentation of mixtures should be the sum 
of the rates for the constituents. This, as shown above, is not found 
to be the case, and it is therefore necessary to suppose, either that one 
sugar influences the fermentation of another in some unknown way, 
or that only a part of the mechanism of fermentation is specific for the 
particular sugar. Thus the enzyme may be specific and the co-enzyme 



THE MECHANISM OF FERMENTATION 135 

non-specific, so that only a certain maximum rate is attainable, or 
again, the supply of free phosphate may be the controlling factor. 

In the prevailing state of ignorance as to the exact function of the 
co-enzyme and of the conditions upon which the velocity of ferment- 
ation in the cell depends, it is at present impossible to decide between 
these various theories, but they all offer points of attack which justify 
the hope that much further information can be obtained by experi- 
mental inquiry. 

It will be seen from the foregoing that Buchner's discovery of zymase 
has opened a chapter in the history of alcoholic fermentation which is 
yet far from being completed. In every direction fresh problems 
present themselves, and it cannot be doubted that as in the past, the 
investigation of the action of the yeast-cell will still prove to be of 
fundamental importance for our knowledge of the mode in which 
chemical change is brought about by living organisms. 



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INDEX. 



ACETALDEHYDE, as an intermediate product 
of alcoholic fermentation, no. 

reduction of by yeast, no. 
Acetone-yeast, 38. 

Alanine, as an intermediate product of alco- 
holic fermentation, 115. 
Alcohol, formation of, from sugar by alkalis 

97- 

Alcoholic fermentation, attempts to separate 
enzymes of, from yeast-cell, 15. 

by-products of, 85. 

equation of, 51. 

Gay-Lussac's theory of, 4. 

Iwanoff's theory of, 106. 

kinetics of, 120, 128. 

Lavoisier's views on, 3. 

Liebig's theory of, 8. 

Nageli's theory of, 15. 

of the amino-acids, 87. 

theory of, 91. 

Pasteur's researches on, n. 

Traube's enzyme theory of, 14. 

Alkalis, effect of, on hexoses, 96. 
Amino-acids, alcoholic fermentation of, 87. 

stereoisomerides of, fermented at differ- 

ent rates by yeast, 89. 
d-Amyl alcohol, formation of from isoleuc- 

ine, 86. 

Antiprotease in yeast-juice, 42, 65. 
Antiseptics, action of, on yeast-juice, 19, 36. 
Arsenate, effect of, on fermentation by 

yeast-juice and zymin, 75. 
on autofermentation of yeast-juice, 80. 

nature of acceleration produced by, 78. 
Arsenite, effect of, on fermentation by yeast- 
juice, 77. 

on autofermentation of yeast-juice, 80. 

nature of acceleration produced by, 78. 
Autofermentation of yeast-juice, 33, 119. 

effect of arsenates and arsenites on, 80. 

BAEYER'S theory of fermentation, 99. 
Boiled yeast-juice, effect of, on fermentation 
by yeast-juice, 41. 

CARBOXYLASE, 81, 93. 

relation of to alcoholic fermentation, 83. 
Co-enzyme, effect of electric current on, 67. 

enzymic destruction of, 63. 

of yeast-juice, 59. 

precipitation of, by ferric hydroxide, 67. 

properties of, 63. 

removal of, from yeast-juice, 59. 

separation from phosphate and hexose- 

phosphate, 67. 

Concentration of sugar, effect of, on fermen- 
tation by yeast-juice, 34. 



DAUERHEFE, 38. 

Diastatic enzyme of yeast-juice, 33. 

Dihydroxyacetone, fermentability of, 104. 

formation of, in fermentation, 105. 
Dried yeast (Lebedeff), 24, 38. 

ENDOTRYPTASE, 20. 
Enzyme action, laws of, 121. 
Enzymes, combined with protoplasm, 126. 
Equation of alcoholic fermentation, 51. 

FERMENTATION by yeast-juice, causes of 

cessation of, 64. 
Fermenting complex, 63. 

power of yeast-juice, estimation of, 27. 
Formaldehyde, production of in alcoholic 

fermentation, 117. 

Formic acid theory of fermentation, 114. 
Fructose, fermentation of, by yeast-juice, 32. 
in presence of phosphate, 73. 

relation of, to fermenting complex, 74. 
Fusel oil, formation of, from amino-acids, 85. 

GALACTOSE, fermentation of, by yeast, 131. 

fermentation of, by yeast-juice, 32. 
Glucose, fermentation of, by yeast-juice, 32. 
Glyceraldehyde, fermentability of, 104. 
Glyceric acid, fermentation of, 108. 
Glycerol, formation in fermentation, 95. 
Glycogen as an intermediate product of 

alcoholic fermentation, 116. 

fermentation of, by yeast-juice, 33. 

removal of, from yeast, 39. 
Grinding of yeast by hand, 22. 

mechanical, 23. 

Glutamic acid, decomposition of, by yeast, 
90. 

HEFANOL, 38. 

Hexosediphosphoric acid phenylhydrazone, 
hydrazine salt of, 50. 

Hexosemonophosphoric acid osazone, hydra- 
zine salt of, 50. 

Hexosephosphatase, 54. 

effect of arsenate and arsenite on action 

of, 79- 
Hexosephosphate, constitution of, 51. 

enzymic decomposition of, in yeast- 

juice, 56. 
hydrolysis of, 51. 

formation of, 48. 

hydrolysis of, by acids, 49. 

preparation of, 48. 

properties of, 49. 

theory of formation of, 57, 117, 
Hexoses, action of alkalis on, 96. 



155 



5 6 



INDEX 



ISO-AMYL alcohol, formation from leucine, 87. 
Isoleucine, decomposition of, by yeast, 87. 

O-KETONIC acids, fermentation of, 81. 

LACTIC acid, destruction of, by yeast-juice, 
102. 

formation from sugars by alkalis, 97. 

of, in yeast-juice, 102. 

non-fermentability of, by yeast, 103. 

theory of fermentation, 102. 

Leucine, decomposition of, by yeast, 87. 

MACERATION extract, preparation of, 25. 
Mannose, fermentation of, by yeast, 131. 

of by yeast -juice, 32. 

Methylglyoxal, conversion of, into lactic 
acid, 101. 

non-fermentability of, 104. 

as an intermediate product of alcoholic 

fermentation, 113. 

OXALACETIC acid, formation of, from tartaric 
acid, 101. 

PERMANENT yeast, 38. 
Phenylethyl alcohol, 88. 
Phosphate, changes of, in alcoholic fermen- 
tation, 47. 

effect of, on fermentation by yeast-juice, 

42. 

by zymin, 46. 

of fructose, 73. 

of on total fermentation of yeast- 
juice, 54. 

influence on fermentation of concentra- 

tion of, 71. 

inhibition by, 71. 

Phosphates, essential for alcoholic fermenta- 
tion, 55. 

Proteoclastic enzyme of yeast, 20. 

Protoplasmic theory of activity of yeast- 
juice, 19. 

Pyruvic acid, fermentation of, 81. 
theory of fermentation, 109. 

RATE of fermentation, controlling factors of, 
119. 

Reductase, intervention of, in alcoholic fer- 
mentation, in. 

SERUM, effect of, on fermentation by yeast- 
juice, 41. 

Succinic acid, formation of, in fermentation, 
89. 

formed from glutamic acid by yeast, 

90. 

Synthetic enzyme in yeast-juice, 32. 



TEMPERATURE coefficient of fermentation 

by yeast, 129. 

by zymin, 122. 

esterification of phosphoric acid 

by yeast extract, 58. 
Tryptophol, 88. 
Tyrosol, 88. 

WOHL'S theory of fermentation, 101. 

YEAST, action of toluene on, 124. 

and yeast-juice, fermentation by, com- 

pared, 29, 124. 

discovery of the vegetable nature of, 5. 

fermentation by, 127. 

of different sugars by, 130. 

influence of concentration of dextrose 

on fermentation by, 128. 
of, on rate of fermentation, 

129. 
of toluene on autofermentation of, 

126. 

nature of the process of fermentation 

by, 123. 

temperature coefficient of fermentation 

by, 129. 

theories of fermentation by, 133. 
Yeast-juice and yeast, fermenting powers 

compared, 29, 124. 

co-enzyme of, 59. 

dialysis of, 59, 62. 

effect of arsenate on fermentation by, 75. 
of concentration of sugar on fermen- 
tation by, 34. 

-of dilution on fermentation by, 35. 
of phosphate on total fermentation, 

produced by, 54. 

estimation of fermenting power of, 27. 

evaporation of, 37. 

filtration of through gelatin filter, 59. 

precipitation of, 38. 

preparation of, 21. 

properties of, 19. 

ratio of alcohol and carbon dioxide, 

produced by, 30. 

synthesis of complex carbohydrate by, 

3 1 - 

variation of rate of fermentation by, 

with concentration of sugar, 121. 

ZYMASE, Buchner's discovery of, 16. 

enzymic destruction of, 64. 

properties of, 18. 

regeneration of inactive, 64. 

separation from co-enzyme, 59. 
Zymin, 21, 38. 

fermentation by, 39. 

rate of fermentation by, 39. 

temperature coefficient ofi fermentation 

by, 122. 



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